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Biomedical subjects

M Takagi

Publications and source records attributed to M Takagi.

At least 217 records · Page 12Linked to original sources
PubMed1999

Spontaneously developed aneurysm of the ductus arteriosus in an adult.

We report an unusual adult case of aneurysm associated with the ductus arteriosus. A 67-year-old female, who suffered recurrent nerve palsy, was diagnosed as having an aneurysm of the ductus arteriosus. The aneurysm, located in the medial aspect of the aortic ischimus and projected toward the pulmonary artery, was successfully repaired using a Dacron patch. A pathologic review of the aneurysm revealed the degeneration of the aortic media.

Aged↗
PubMed1999

Unfolded protein response-induced BiP/Kar2p production protects cell growth against accumulation of misfolded protein aggregates in the yeast endoplasmic reticulum.

Overproduction of delta(pro), a mutated secretory proteinase derived from a filamentous fungus Rhizopus niveus, results in formation of gross aggregates (delta(pro) aggregates) in the yeast endoplasmic reticulum (ER) lumen, activation of the unfolded protein response (UPR) and ER membrane proliferation. To investigate the roles of the UPR against the delta(pro) aggregates, we constructed an IRE1-deleted ((delta)ire1) strain. In contrast to wild-type cells, (delta)ire1 cells ceased to grow several hours after the overproduction of (delta)pro. Two lines of evidence argued against the possibility that the growth defect was due to the inability to make extra ER membrane which accommodates the (delta)pro aggregates. First, by electron microscopy, ER membrane proliferation was observed in (delta)ire1 cells overproducing (delta)pro. Second, disruption of the OPI1 gene in the (delta)ire1 mutant, which is considered to derepress the activities of phospholipid-synthesizing enzymes, did not restore the growth upon the overproduction of (delta)pro. Instead, the growth was restored when an extra copy of the KAR2 gene, which encodes yeast BiP, was introduced, indicating that an increase in the amount of BiP is essential for cell growth when the (delta)pro aggregates accumulate in the ER. Since BiP is included in the insoluble (delta)pro aggregates, it is likely that the amount of free BiP in the ER lumen is insufficient without the UPR to fully exert its functions. Consistently, overproduction of (delta)pro impaired protein translocation and folding in (delta)ire1 cells but not in wild-type cells. The tunicamycin sensitivity of (delta)ire1 cells was also suppressed by extra expression of KAR2, suggesting that BiP plays a principal role in protecting cell growth against misfolded proteins accumulated in the ER.

Base Sequence↗
PubMed1999

Rapid method for detection and detoxification of heavy metal ions in water environments using phytochelation.

Phytochelatins (PCs, (gammaGlu-Cys)n-Gly (n = 2-11)) are produced by higher plants, algae, and some fungi in response to heavy metal ion exposure. A rapid and convenient method for quantifying heavy metal ion concentrations in water environments was developed using a chemically synthesized PC as a mediator. The chelating ability of the PC and quantification of the thiol group were utilized to measure heavy metal ions at low concentrations. The method requires only ten minutes for measurement and only 1 ml of a liquid sample. A range of homogeneous PCs (n = 4-7) were chemically synthesized using a peptide synthesizer. These, especially PC7, exhibited higher sensitivity and consistency of measurement than the native PC from Silene cucubalus, which produced a mixture of PC2, PC3, and PC4. Detoxification of heavy metal ions in vitro by PC was also investigated. Using the paper disc method, the cell growth inhibition zone caused by cadmium ion against Salmonella typhimurium TA1538 was significantly decreased by addition of PC. Furthermore, at the minimum inhibitory concentration of cadmium ion (200 microM) in a nutrient broth culture of S. typhimurium, cell growth was almost completely recovered by addition of PC to the medium.

Journal Article↗
PubMed1999

Starch particles modified with gelatin as novel small carriers for mammalian cells.

Promotion of cell aggregation by addition of small starch particles with and without modification with gelatin was investigated, and the influence of CHO cell aggregation by the addition of modified starch on substrate consumption and production of tissue plasminogen activator (tPA) was investigated. Addition of starch particles (approximately 10 micromPhi) covalently bonded with gelatin was very effective in forming cell aggregates ranging from 100 to 300 microm in diameter and decreased the cell sedimentation time to one-thirtieth that of the normal time. There was almost no influence of the addition of modified starch particles on cell metabolism and tPA production. Cell aggregation makes it easy to separate cells from culture during changing of medium in a cell suspension culture without the use of special equipment, such as centrifuges, hollow fiber membranes, and spin filters.

Journal Article↗
PubMed1999

Gene analysis and enzymatic properties of thermostable beta-glycosidase from Pyrococcus kodakaraensis KOD1.

A beta-glycosidase with broad substrate specificity was identified from a hyperthermophilic archaeon, Pyrococcus kodakaraensis KOD1. The gene encoding beta-glycosidase (Pk-gly) consists of 1449 nucleotides corresponding to a polypeptide of 483 amino acids. The protein showed similarity with other beta-glycosidases from family-1 glycosyl hydrolases, in particular, it showed high identity to beta-mannosidase from P. furiosus (55.7%), beta-glycosidase from Sulfolobus solfataricus (42.7%) and beta-glucosidase from P. furiosus (41.9%). The cloned gene was expressed in Escherichia coli and the recombinant protein was purified. The beta-glycosidase showed optimal activity at pH 6.5 and at an extremely high temperature of 100 degrees C, and had a half-life of 18 h at 90 degrees C. The beta-glycosidase hydrolyzed various pNp-beta-glycopyranosides, with kcat K(m) values in the order of pNp-beta-glucopyranoside = pNp-beta-mannopyranoside > pNp-beta-galactopyranoside > pNp-beta-xylopyranoside. pNp-beta-mannopyranoside was the substrate exhibiting the lowest K(m) value [0.254 mM] with a kcat K(m) ratio comparable to that of pNp-beta-glucopyranoside. This substrate specificity was distinct from previously reported beta-glycosidases. We observed that the region in PK-Gly corresponding to the fifth alpha-helix and beta-strand region of beta-glycosidase from S. solfataricus, which constitutes a large portion of the channel for substrate incorporation, displayed a chimeric structure, with the N-terminal region similar to beta-glycosidases and the C-terminal region similar to beta-mannosidases. An exo-type hydrolytic activity and transglycosylation activity were also observed towards cellooligomers.

Journal Article↗
PubMed1999

Effect of temperature on cell population balance in Dexter's culture of murine bone marrow hematopoietic cells with stromal cells.

The effect of temperature in the range from 25 to 37 degrees C on the population balance of stromal and multiple-lineage hematopoietic cells from murine bone marrow at various stages of differentiation in Dexter's culture was investigated. The length of time required for stromal cells to reach confluence after inoculation of both cell types harvested from murine bone marrow was shorter at higher temperatures. On the other hand, the hematopoietic cell concentration initially decreased more rapidly at higher temperatures until the confluence of stromal cells, which should then support the proliferation of hematopoietic cells. The concentration of hematopoietic cells began to increase after 2 weeks incubation at 33 and 37 degrees C and after 4 weeks at 29 degrees C. However, the growth of hematopoietic cells was not stable at 37 degrees C, and neither stromal nor hematopoietic cells showed significant growth at 25 degrees C. The specific growth rate of hematopoietic cells at 29 degrees C after 4 weeks was comparable or higher than that at 33 degrees C, while the final concentration of hematopoietic cells was maximal at 33 degrees C. Using a cultivation method in which hematopoietic cells were recharged onto a confluent layer of stromal cells prepared at 33 degrees C, the maintenance and growth of hematopoietic cells were better at 29 degrees C than at higher temperatures. The content of progenitor cells among the hematopoietic cells increased prior to the increase in the hematopoietic cell concentration, and the progenitor cell was greater content at the lower temperature. These results suggest that cultivation at 29 degrees C might be superior to 33 degrees C for the maintenance and growth of hematopoietic cells with a prepared confluent layer of stromal cells.

Journal Article↗
PubMed1999

Metabolic flux change in hybridoma cells under high osmotic pressure.

The effect of high osmotic pressure on the flux changes of two energy metabolisms from glucose and glutamine by AFP-27 hybridoma cells producing monoclonal antibody (MAb) was investigated in batch cultures at various osmotic pressures in the range from 300 to 424 mOsmol/kg. The specific production rate of MAb (q(MAb)) increased monotonically with increasing osmotic pressure. The specific consumption rates of glucose (nu(G)) and glutamine (nu(GLN)) also increased with the osmotic pressure up to 410 mOsmol/kg. However, there were marked changes in the metabolic fluxes when the osmotic pressure was raised further, resulting in a decrease in nu(G) while nu(GLN) continued to increase. These changes in the metabolic fluxes at osmotic pressures higher than 410 mOsmol/kg were associated with an increased yield of lactic acid from glucose (Y (L G )), which indicated the energy yield from glucose declined at osmolarities higher than 410 mOsmol/kg. On the other hand, because of a larger increment in the specific production rate of ammonia under high osmotic pressure, the yield of ammonia from glutamine (Y (A GLN )) increased monotonically with the osmotic pressure throughout the range 300 to 424 mOsmol/kg, signifying a rise in the energy yield from glutamine. Consequently, the higher specific ATP production rates from glucose and glutamine associated with the flux changes under high osmotic pressure could be one of the reasons for the increase in q(MAb) observed at high osmotic pressures.

Journal Article↗
PubMed1999

Effects of bone morphogenetic protein-2 and transforming growth factor-beta1 on gene expression of decorin and biglycan by cultured osteoblastic cells.

The influence of bone morphogenetic protein-2 (BMP-2) and transforming growth factor beta (TGF-beta) on the expression of small proteoglycans, decorin and biglycan was investigated in a clonal rat osteoblastic cell line, ROS-C26 (C26) cells, which is a potential osteoblast precursor cell line and capable of differentiating into mature osteoblasts after treatment with recombinant BMP-2 (rhBMP-2). Following the culture of C26 cells for 3, 6, and 9 days in the presence or absence of rhBMP-2, alkaline phosphatase activity increased in the rhBMP-2 treated cells in direct proportion to their differentiation into more mature osteoblastic cells, whereas decorin mRNA decreased in the cells, when compared to control cells without rhBMP-2 treatment. These results were evident 6 days after treatment. However, rhBMP-2 treatment had no effect on biglycan mRNA expression in the cells. Subsequently, after removal of rhBMP-2 from the culture media, the cells were further cultured for 24 h with graded concentrations of TGF-beta1 (0, 0.1, 1.0, 5.0, and 10 ng/ml). TGF-beta1 decreased decorin mRNA expression in the cells dose dependently, but did not affect their biglycan mRNA expression. Furthermore, either removal of rhBMP-2 from the culture media or addition of TGF-beta1 significantly decreased alkaline phosphatase activity of rhBMP-2-induced cells. These results indicate that osteoblastic differentiation is accompanied by increased alkaline phosphatase activity and decreased expression of decorin mRNA, but continuous expression of biglycan mRNA. Both rhBMP-2 and TGF-beta1 inhibit decorin mRNA expression in osteoblasts at varying stages of differentiation, but their effects on biglycan mRNA expression and alkaline phosphatase are different.

Alkaline Phosphatase↗
PubMed1999

Effects of transforming growth factor-beta1 on the gene expression of decorin, biglycan, and alkaline phosphatase in osteoblast precursor cells and more differentiated osteoblast cells.

In this study, the effects of incubating two clonal rat osteoblastic cell lines at different stages of differentiation, ROB-C26 (C26) and ROB-C20 (C20), with transforming growth factor-beta1 (TGF-beta1) on the gene expression of decorin, biglycan, and alkaline phosphatase were examined. C26 cells are a potential osteoblast precursor cell line that is also capable of differentiating into muscle cells and adipocytes and is differentiated into osteoblasts after treatment with bone morphogenetic protein-2. C20 cells are a more differentiated osteoblastic cell line. Our Northern blot studies demonstrated that after treatment with TGF-beta1 (0, 0.1, 1.0, 5.0, and 10 ng/ml), a dose- and time-dependent decrease in decorin mRNA expression was found in C26 cells. In contrast, the effect of decorin mRNA with TGF-beta1 was not determined in C20 cells, since decorin mRNA expression was extremely low in this cell line even in the absence or presence of TGF-beta1. Although TGF-beta1 treatment resulted in no appreciable effect on biglycan mRNA expression in both cell lines in a dose- and time-dependent manner, it decreased significantly the expression of alkaline phosphatase in both cell lines at the gene and protein level. Reverse transcriptase-polymerase chain reaction analysis revealed the gene expression of decorin, and TGF-beta type I and type II receptors in both cell lines. These results indicate that osteoblasts progenitor cells express both decorin and biglycan mRNAs. In contrast, more differentiated and mature osteoblastic cells express preferentially biglycan mRNA. TGF-beta1 exerts different effects on the expression of decorin and biglycan mRNAs, and is a potent inhibitor of the gene expression of alkaline phosphatase during osteoblast differentiation.

Alkaline Phosphatase↗
PubMed1999

Sequence variations of Epstein-Barr virus LMP2A gene in gastric carcinoma in Japan.

The latent EBV gene products expressed in Epstein-Barr virus (EBV)-associated gastric carcinoma (EBVaGC), are only LMP2A, EBNA-1, BARF-0 and EBERs. To examine the correlation between LMP2A sequence variation in EBVaGC and transformation of the cells, the complete sequence of the LMP2A gene was determined in three cases of Japanese EBVaGC and compared with the prototype B95-8 strain. In addition, the sequences of exons 2,6 and 7 of LMP2A were determined in four to six EBVaGC cases. The results of sequence analysis indicated that LMP2A of EBVaGC was structurally very similar to B95-8, but contained a significant nucleotide variation. Ten nucleotide substitutions were identified in almost all cases tested, and three of these caused amino acid changes. Of these three, two amino acid substitutions were not expected to change any known functions of LMP2A. The other amino acid substitution from serine to threonine was located at codon 348 within one of the target epitopes of EBV-specific cytotoxic T-lymphocytes. The LMP2A of EBV in peripheral blood lymphocytes from six healthy individuals showed serine (4/6 cases) or threonine (2/6 cases) substitution at codon 348, while LMP2A with the threonine substitution was the major form (5/6 cases) observed in EBVaGC, indicating that EBV with the threonine substitution may confer an advantage for viral persistence in tumor cells. However, our sequencing results suggested that the LMP2A protein in EBVaGC is functionally similar to that of the B95-8 strain and is not unique to gastric carcinoma, indicating the importance of LMP2A for EBV latency.

Amino Acids↗
PubMed1999

Complete analysis of genes and enzymes for gamma-hexachlorocyclohexane degradation in Sphingomonas paucimobilis UT26.

gamma-Hexachlorocyclohexane (gamma-HCH; also called BHC or lindane) is one of the highly chlorinated pesticides which can cause serious environmental problems. Sphingomonas paucimobilis UT26 degrades gamma-HCH under aerobic conditions. The unique degradation pathway of gamma-HCH in UT26 is revealed. In the upstream pathway, gamma-HCH is transformed to 2,5-dichlorohydroquinone (2,5-DCHQ) by two different dehalogenases (LinA and LinB) and one dehydrogenase (LinC) which are expressed constitutively. In the downstream pathway, 2,5-DCHQ is reductively dehalogenated, and then ring-cleaved by enzymes (LinD and LinE, respectively) whose expressions are regulated. We have cloned and sequenced five structural genes (linA, linB, linC, linD, and linE) directly involved in this degradation pathway. The linD and linE genes form an operon, and its expression is positively regulated by the LysR-type transcriptional regulator (LinR). The genes linA, linB, and linC are constitutively expressed, and are present separately from each other in the UT26 genome. Cell fractionation analysis, Western blotting, and immuno electron microscopy revealed that LinA and LinB are localized in the periplasmic space of UT26.

Journal Article↗
PubMed1999

DNA damage-associated cell cycle and cell death control is differentially modulated by caffeine in clones with p53 mutations.

Caffeine is known to potentiate the cytotoxic effects of DNA damaging agents and increases the sensitivity of p53-deficient cells to X-irradiation (X-IR). We have analyzed the cell cycle and cell death control after X-IR in the absence or presence of caffeine in hematological cell lines with various configurations of the p53 gene; EBV-immortalized lymphoblastoid cells with heterozygous p53 mutation (wt/mt), human leukemia cell lines HL60 and KOPM28 with no and mutant p53 expression, respectively. These cell lines display an impaired G0/G1 checkpoint and G2 delay following X-IR, and resistance to apoptosis, which are in accordance with findings previously reported. When irradiated in combination with caffeine, all these cell lines overrode the G2 delay and accumulated at G0/G1. The cell cycle modifications in these cell lines correlated with the increase in radiation-induced p34Cdc2 kinase activity by caffeine. These cell cycle control modifications by caffeine, however, were not associated with enhancement of radiation-induced apoptosis or reduction of clonogenic growth activity in these cell lines. These results suggest that the cytocidal effect of caffeine may need to be verified independently of its cell cycle regulatory activities at least in some cases with p53 mutation.

Amino Acid Substitution↗
PubMed1999

Role of free radicals in the pathogenesis of lipid-induced glomerulosclerosis in rats.

BACKGROUND: We examined whether a high-cholesterol (HC) diet causes glomerulosclerosis in rats, and investigated the role of free radicals and lipid peroxidation in lipid-induced glomerulosclerosis. METHODS: The rats were given a normal diet, a HC diet, or a HC diet with antioxidants and radical scavengers. Serum levels of lipid, lipid peroxide (LOOH), urinary excretion of protein (UP), and urinary norepinephrine excretion (UNE) were measured. The glomerular sclerosing score was used to evaluate the renal injury. RESULTS: Blood pressure, total cholesterol, and LOOH were increased by a HC diet, as were UP and UNE. The HC diet induced renal injury. Treatment with superoxide dismutase, dimetylthiourea as a scavenger of hydroxyl radical (OH.), defferoxamine masilate as an iron chelator, or vitamin E inhibited the increases in blood pressure, LOOH, UP, and UNE, whereas total cholesterol was not affected. The production of superoxide anion (O2-.) by neutrophil and LOOH in the kidney was increased, and superoxide dismutase and hydrogen peroxide in the kidney were decreased. Almost all of these changes were attenuated by vitamin E; however, the O2-. production was not inhibited. OH. was increased by the HC diet, and it was normalized with the treatments. Furthermore, the sclerosing score was partially suppressed by the treatments. Ferric iron was stained in the proximal tubulus, and it was not observed in the treated rats. CONCLUSIONS: The data suggest that lipid peroxidation is involved in the pathogenesis of lipid-induced glomerulosclerosis and that O2-. and OH. may play a role in the process.

Animals↗
PubMed1999

Chimeric cytokine receptor can transduce expansion signals in interleukin 6 receptor alpha (IL-6Ralpha)-, IL-11Ralpha-, and gp130-low to -negative primitive hematopoietic progenitors.

We generated transgenic mice expressing chimeric receptors, which comprise extracellular domains of the human granulocyte-macrophage colony-stimulating factor (hGM-CSF) receptor and transmembrane and cytoplasmic domains of the mouse leukemia inhibitory factor receptor. In suspension cultures of lineage-negative (Lin(-)), 5-fluorouracil-resistant bone marrow cells of the transgenic mice, a combination of hGM-CSF and stem cell factor (SCF) induced exponential expansions of mixed colony-forming unit. The combination of hGM-CSF and SCF was effective on enriched, Lin(-)Sca-1(+)c-kit(+) progenitors and increased either mixed colony-forming unit or cobblestone area-forming cells. In case of stimulation with hGM-CSF and SCF, interleukin-6 (IL-6) and SCF, or IL-11 and SCF, the most efficient expansion was achieved with hGM-CSF and SCF. When Lin(-)Sca-1(+)c-kit(+)CD34(-) further enriched progenitors were clone sorted and individually incubated in the presence of SCF, hGM-CSF stimulated a larger number of cells than did IL-6, IL-6 and soluble IL-6 receptor (IL-6R), or IL-11. These data suggest the presence of IL-6Ralpha-, IL-11Ralpha-, and gp130-low to -negative primitive hematopoietic progenitors. Such primitive progenitors are equipped with signal transduction molecules and can expand when these chimeric receptors are genetically introduced into the cells and stimulated with hGM-CSF in the presence of SCF.

Animals↗
PubMed1999

Mark-release-recapture experiment with adult Anopheles minimus (Diptera: Culicidae) on Ishigaki Island, Ryukyu Archipelago, Japan.

Movement of adult An. minimus Theobald between larval habitats and feeding sites was investigated by mark-release-recapture in March 1993 on Ishigaki Island, Ryukyu Archipelago, Japan. Unfed (998 females) and fed (1,485 females) cohorts from a laboratory colony were marked with different colors of fluorescent dye, and released near Nishihama stream where larvae were abundant. Overall, 42 fed (2.82%) and 13 unfed (1.30%) females were recaptured. Among these, 12 females were recaptured at a cowshed, the nearest blood meal source. Most females in the fed cohort recaptured at the release point had developing or matured ovaries, whereas 55% at the cowshed were parous. The movement of released females from larval habitat to the cowshed for blood feeding indicated a flight distance > 1 km.

Animals↗
PubMed1999

DNA coated with cationic fullerene derivative. A possible microwire in water.

Fullerene derivative 2 carrying pyridinium cation bound to sonicated calf thymus DNA in water. The binding ratio was 1 fullerene unit to 1 phosphate residue, giving the complex where DNA strand is seemingly coated with electron-conducting fullerenes. Cyclic voltammetry shows three-step redox couples in the complex, and the current peaks were broadened and shifted to positive side as compared to uncomplexed 2. Binding of 2 onto grooves of DNA double helix was suggested.

Carbon↗
PubMed1999

Metal ion-directed DNA recognition by chelating DNA ligands.

Novel DNA ligand bearing oxine and pyridinium group has been synthesized. The DNA binding of this ligand was regulated by coexisting Cu2+ over the range of more than three orders in magnitude of binding constant. This should be due to the metal-mediated dimerization of the ligand and concomitant emergence of cationic charges. There were definite two DNA binding modes for the ligand dimer depending on the P/L ratio.

Chelating Agents↗