PubMed Health⌕ Search

Biomedical subjects

M Takai

Publications and source records attributed to M Takai.

At least 37 records · Page 2Linked to original sources
PubMed1999

Involvement of cytochrome a in iron oxidation of a moderately thermophilic iron-oxidizing bacterium, strain TI-1.

The iron-oxidizing activity of a moderately thermophilic iron-oxidizing bacterium, strain TI-1, was located in the plasma membrane. When the strain was grown in Fe2+ (60 mM)-salts medium containing yeast extract (0.03%), the plasma membrane had iron-oxidizing activity of 0.129 mumol O2 uptake/mg/min. Iron oxidase was solubilized from the plasma membrane with 1.0% n-octyl-beta-D-glucopyranoside (OGL) containing 25% (v/v) glycerol (pH 3.0) and purified 37-fold by a SP Sepharose FF column chromatography. Iron oxidase solubilized from the plasma membrane was stable at pH 3.0, but quite unstable in the buffer with the pH above 6.0 or below 1.0. The optimum pH and temperature for iron oxidation were 3.0 and 55 degrees C, respectively. Solubilized enzyme from the membrane showed absorption peaks characteristic of cytochromes a and b. Cyanide and azide, inhibitors of cytochrome c oxidase, completely inhibited iron-oxidizing activity at 100 microM, but antimycin A, 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO) and myxothiazol, inhibitors of electron transport systems involved with cytochrome b, did not inhibit enzyme activity at 10 microM. The absorption spectrum of the most active enzyme fraction from SP Sepharose FF column chromatography (4.76 mumol O2 uptake/mg/min) compared with lower active fractions from the chromatography (0.009 and 2.10 mumol O2 uptake/mg/min) showed a large alpha-peak of cytochrome a at 602 nm and a smaller alpha-peak of cytochrome b at 560 nm. The absorption spectrum of pyridine ferrohemochrome prepared from the most highly purified enzyme showed an alpha-peak characteristic of heme a at 587 nm, but not the alpha-peak characteristic of heme c at 550 nm. The cytochrome a, but not cytochrome b, in the most highly purified enzyme fraction was reduced by the addition of ferrous iron at pH 3.0, indicating that electrons from Fe2+ were transported to cytochrome a, but not cytochrome b. These results strongly suggest that cytochrome a, but not cytochromes b and c, is involved in iron oxidation of strain TI-1.

Animals↗
PubMed1998

Reactive oxygen species generated from the reaction of copper(II) complexes with biological reductants cause DNA strand scission.

The generation of hydroxyl radicals (.OH) from the reaction of Cu(II) complexes with biological reductants such as ascorbic acid, glutathione, acetylcysteine, and hydroquinone was confirmed by spin-trapping experiments using electron spin resonance (ESR). The following Cu(II) complexes were used: Cu(II)-(CyHH)2 (CyHH, cyclo(L-histidyl-L-histidyl)), Cu(II)(OP)2 (OP, o-phenanthroline), Cu(II)(HGG) (HGG, L-histidyl-glycylglycine), and Cu(II)(en)2 (en, ethylenediamine). The methyl radical adduct of alpha-(pyridyl-4-N-oxide)-N-tert-butylnitrone (POBN-CH3) was obtained from the reaction of ascorbic acid with all Cu(II) complexes used here in the presence of a spin trap, POBN, and dimethyl sulfoxide, indicating the generation of .OH. Glutathione, N-acetylcysteine, and hydroquinone reacted with both Cu(II)(CyHH)2 and Cu(II)(OP)2 to generate POBN-CH3, while these reductants did not react with either Cu(II)(HGG) or Cu(II)(en)2. Interestingly, the formation of POBN-CH3 in the reaction of Cu(II)-(CyHH)2 with glutathione or N-acetylcysteine was found only at a Cu(II)(CyHH)2/glutathione or Cu(II)(CyHH)2/N-acetylcysteine ratio of 1. The DNA strand scission caused by reaction mixtures of Cu(II) complexes with reductants was investigated under the same conditions as the ESR spin-trapping experiments. Addition of ascorbic acid to mixtures of these four Cu(II) complexes and DNA resulted in DNA strand breakage. Hydroquinone plus Cu(II)(CyHH)2 also caused DNA strand scission. In addition, DNA strand breakage was observed with the reaction of Cu(II)(OP)2 with glutathione, N-acetylcysteine, and hydroquinone. In contrast, reaction mixtures of glutathione, N-acetylcysteine, or hydroquinone with Cu(II)-(HGG) or Cu(II)(en)2 did not cause DNA strand scission within the concentration range used. The results obtained here suggest that there is a good correlation between POBN-CH3 formation and DNA strand scission. Thus, DNA strand scission may be caused by .OH generated from the reaction of some Cu(II) complexes with biological reductants under aerobic conditions. Since ascorbic acid, glutathione, and N-acetylcysteine are present in living cells, some Cu(II) complexes may be capable of initiating DNA damage in the presence of these reductants.

Acetylcysteine↗
PubMed1998

Structural study on silkworm eclosion hormone fragment (1-34) in solution by proton nuclear magnetic resonance spectroscopy.

Eclosion hormone (EH) is a neuropeptide hormone which controls the ecdysis behavior in insect. The three dimensional structure of the N-terminal fragment (1-34) of the eclosion hormone which was predicted to contain a compact region crucial for the EH activity was studied in 50% d3-trifluoroethanol(TFE)/50% H2O at pH 3 and 298 K by 1H NMR spectroscopy with the combined use of distance geometry and molecular dynamics calculations. NMR results indicated that the fragment actually assumes an alpha-helix between Ala10 and Gln20, but no rigid structure is present from Cys21 through the C-terminus and for the N-terminal region (Ser1-Asp9). The elucidated structure was compared with the predicted structure of the native EH for the further development of the design of the insecticide.

Amino Acid Sequence↗
PubMed1998

Structural studies on endothelin receptor subtype B specific agonist IRL 1620 [suc-[Glu9, Ala11,15]ET-1(8-21)] and its analogs with dipalmitoyl phosphatidylcholine vesicles by NMR spectroscopy.

IRL 1620 ¿suc-[Glu9,Ala11,15]ET-1(8-21)¿ is a potent and specific agonist for the ET(B) receptor. Five analogs of IRL 1620 were synthesized in this study. These were all C-terminal linear peptides of endothelin 1 (ET-1) comprising 14 amino acid residues and exhibiting highly potent ET(B) receptor binding affinities. The peptides consisted of three pairs and each component of the pairs differed from its partner in only the 18th residue, i.e. Asp was replaced by Gly. The replacements resulted in more than a 10-fold increase in affinity to the ET(A) receptor. The structures of these peptides were investigated in the presence of phospholipid vesicles (dipalmitoyl phosphatidylcholine) by NMR spectroscopy. By the replacement of Asp by a less bulky Gly, the C-terminal tripeptide region folded back toward the helical region, making it shorter than the Asp-substituted peptide helical region. Such a folded conformational feature may explain the increased binding affinity to ET(A) receptor.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1998

Purification and characterization of sulfur reductase from a moderately thermophilic bacterial strain, TI-1, that oxidizes iron.

A moderately thermophilic bacterium, strain TI-1, produces H2S outside of the cells when grown at 45 degrees C on Fe(2+)-medium (pH 1.8) containing elemental sulfur and L-glutamic acid. A newly identified sulfur reductase was present in the cytosol of this strain and was purified to an electrophoretically homogeneous state from strain TI-1. The apparent molecular weight of sulfur reductase was 86,000 by gel filtration and 48,000 by SDS-PAGE, so the enzyme was a homodimer. The enzyme was most active at pH 9.0 and 60 to 70 degrees C, and it catalyzed the reduction of 1 mol of elemental sulfur with 1 mol of NADH to give 1 mol of H2S and 1 mol of NAD+. Elemental sulfur was a specific electron acceptor of this enzyme. Thiosulfate, sulfite, and tetrathionate were not electron acceptors, but inhibited sulfur reductase activity. NADPH was not used as an electron donor.

Acid Phosphatase↗
PubMed1997

Comparative study of chicken and human parathyroid hormone-(1-34)-peptides in solution with SDS.

Molecular conformations of chicken [cPTH-(1-34)] and human [hPTH-(1-34)] parathyroid hormone fragments in aqueous solutions with various concentrations of SDS were investigated by CD, fluorescence and NMR spectroscopy techniques. In the presence of SDS, chicken and human PTH-(1-34) adopt an a-helical structure making up 32-38% of all the peptide amino acids. The process of the a-helical formation of these two fragments is considerably different. The CD spectral change of hPTH-(1-34) was characteristic of a monotonous increase in the negative peak at 222 nm with increasing SDS concentrations. However, for cPTH-(1-34) a beta-turn is formed first, followed by alpha-helix formation upon an increase in SDS concentrations. The change of the tryptophan fluorescence spectra of cPTH-(1-34) is well correlated with the changes in CD spectra, suggesting that the side chain of Trp23 is involved in the conformational change from random coil to alpha-helix via beta-turn. The three-dimensional structure of cPTH-(1-34) with SDS micelle was elucidated by 1H-NMR at pH 3.8 and 300 K, with the combined use of distance geometry and restrained molecular dynamics calculations. NMR results indicated that it contains two helices encompassing residues 7-12 and 24-30, respectively. The C-terminal helix in the residue range of 24-30 is amphiphilic, which is stabilized by the hydrophobic interactions among Trp23, Leu24 and Lys27.

Amino Acid Sequence↗
PubMed1997

Functional morphology of the postcranium and locomotor behavior of Neosaimiri fieldsi, a Saimiri-like Middle Miocene platyrrhine.

A number of postcranial specimens of Neosaimiri fieldsi, a Middle Miocene platyrrhine, were discovered in 1988, 1989, and 1990 at La Venta, Colombia. Until recently only three postcranial specimens of this species had been discovered and the present material adds further information about this taxon's postcranial morphology. In overall skeletal dimensions and in postcranial features, Neosaimiri is most similar to Saimiri among extant medium-sized platyrrhines, but differs from Saimiri in having more rugose surface markings, a longer olecranon, a smaller anterior process of the distal tibia, an absence of a distal surface extension on the anterior tibial shaft, an absence of an anterior midtrochlear depression of the talus, and a shorter distal calcaneus relative to the calcaneal tuberosity. These differences suggest that Neosaimiri was relatively heavily built, possessed a more dominant forelimb in quadrupedal progression, and utilized a less stabilized upper ankle joint, and a shorter power arm for plantarflexion. Neosaimiri is interpreted as an arboreal quadruped with frequent leaping across arboreal gaps, as in extant Saimiri, with perhaps less frequent running and leaping than in Saimiri. As with the dentition, the postcranial specimens suggest the close relationship between Neosaimiri and extant Saimiri.

Animals↗
PubMed1997

Primary structures of sperm-specific basic nuclear proteins and gene expression in Japanese newt, Cynops pyrrhogaster.

Electrophoretic analyses of acid extracts from mature sperm of newt, Cynops pyrrhogaster, on acid/urea/Triton X-100 polyacrylamide gel showed the exclusive occurrence of sperm-specific nuclear basic proteins (SBPs), which moved faster than somatic histones on the gel. These SBPs were eluted separately by reversed phase-high-performance liquid chromatography as two large peaks and a few small peaks. Of these, only the small peaks disappeared with treatment of the acid extracts with alkaline phosphatase before they were injected into the column, so that there were only two distinct components: NP1 and NP2. Determination of amino acid sequences by the Edman method as well as by sequencing of cDNA for both components indicated that each protein consisted of 43 (NP1) or 48 (NP2) amino acid residues, rich in arginine residues (53.5% in NP1; 47.9% in NP2), forming the clusters. They had molecular masses of 5,386 Da (NP1) and 5,748 Da (NP2), respectively. Northern blot analysis using cDNAs as probes indicated that mRNAs for both NP1 and NP2 occurred not in primary spermatocytes but in round spermatids. In situ hybridization analyses using antisense RNA for NP1 as a probe clearly showed the first appearance of NP1 mRNA at the late stage of round spermatid.

Amino Acid Sequence↗
PubMed1997

The production of a new water-soluble polysaccharide by Acetobacter xylinum NCI 1005 and its structural analysis by NMR spectroscopy.

A new water-soluble polysaccharide (WSP) was isolated from a culture of Acetobacter xylinum NCI 1005 grown on sucrose. The structure of the WSP was analysed by nuclear magnetic resonance spectroscopy and determined to be a beta-(2-->6)-linked polyfructan, which is structurally different from the polymer synthesized from glucose instead of sucrose by the same strain. The discovery of this new polysaccharide has revealed that the bacterium is able to synthesize two different kinds of water-soluble polysaccharides.

Carbohydrate Sequence↗
PubMed1997

Endothelin evokes efflux of glutamate in cultures of rat astrocytes.

Excessive release of glutamate, from glial cells as well as neurons, is thought to be a major cause of neuronal death in ischemia. To investigate glutamate release from glial cells, we measured glutamate efflux from cultures of rat astrocytes preloaded with L-[3H]glutamate. Glutamate efflux was induced by either 60 mM KCI or Na+-free medium, suggesting that the efflux is due to the reversed operation of a Na+- and K+-coupled glutamate uptake machinery. While investigating various neuropeptides and neurotransmitters, we found that endothelin (ET) specifically induced efflux of glutamate. Northern blot analysis and binding study showed that the ET type B receptor (ET(B)-R) subtype was expressed two to three times more densely than the ET type A receptor (ET(A)-R) in astrocytes. The ET(B)-R antagonist IRL 2500 partially inhibited efflux of glutamate induced by 1 nM ET-1 in a concentration-dependent manner, causing a maximal inhibition of 60% at 1 microM. However, the ET(A)-R antagonist BQ-123 did not cause significant inhibition even at 10 microM. Combination of both antagonists completely inhibited the ET-1-induced efflux. These results indicate that both receptor subtypes are involved in efflux of glutamate with a major contribution from the ET(B)-R. Our findings suggest that ET, which is known to be released in ischemia, may exacerbate neurodegeneration by stimulating efflux of glutamate.

Animals↗
PubMed1997

Prediction of the biologically active sites in eclosion hormone from the silkworm, Bombyx mori.

The structure-activity relationship of eclosion hormone from the silkworm, Bombyx mori, was analyzed. First, the probable active residues in silkworm eclosion hormone and also tobacco hornworm eclosion hormone were predicted by the average distance map method. To examine the contributions of those residues to the activity of silkworm eclosion hormone, Gly-substituted mutants for those predicted residues were produced by site-directed mutagenesis and their activities were evaluated by a bioassay. Finally, Glu12, Met24 and Phe25 were estimated to be the crucial residues for the eclosion hormone activity. The possibility of the development of a blocker of an eclosion hormone receptor on the basis of the present work is also discussed.

Amino Acid Sequence↗
PubMed1996

New specimens of the oldest fossil platyrrhine, Branisella boliviana, from Salla, Bolivia.

During the 1992 and 1993 field seasons, several new specimens of Branisella boliviana, the oldest fossil platyrrhine, were discovered in the late Oligocene deposits at Salla, northwestern Bolivia. The new materials are two maxillary fragments and seven mandibular fragments, including P3 and P2, neither of which have previously been recovered. All new and previously reported materials, including the holotype and referred specimens, were apparently recovered from the same horizon, called the Branisella zone (MacFadden et al. [1985] J. Geol. 93:223-250; MacFadden [1990] J. Hum. Evol. 19:7-21. The crown morphology of the newly discovered upper and lower premolars suggests that Branisella has a close affinity with callitrichines. A detailed examination of all molar specimens suggests that "Szalatavus attricuspis," which was recovered at Salla in 1981 and described as a new genus by Rosenberger et al. (1991), is a junior synonym of Branisella. The Branisella-like upper molar, in which a small hypocone is preserved on the poorly developed distolingual cingulum, is a shared-derived character with callitrichines and does not correspond to the primitive state for platyrrhine phylogeny. The ancestral upper molar morphotype for platyrrhines should have a moderate to large hypocone and a well developed talon area.

Animals↗
PubMed1996

ETA and ETB receptors cooperate in DNA synthesis via opposing regulations of cAMP in human lung cell line.

We investigated the contribution of endothelin type A (ETA) and ETB receptors on ET-induced DNA synthesis in CCD-18Lu cells, a human lung cell line possessing both ETA and ETB (ETA/ETB ratio: 9:1). ET-1 (0.05-2 nM) potently induced [3H]thymidine incorporation by 2- to 14-fold over the basal level. An ETA-selective antagonist, FR139317, inhibited 0.2 nM ET-1-induced DNA synthesis dose dependently, showing complete inhibition at 1 microM. ET-3 was inactive up to 2 nM. In contrast, ETB-selective antagonists, 100 nM of BQ-788 or IRL 2500, partially (30-60%) inhibited 0.2 nM ET-1-induced DNA synthesis. Stimulation of either ETA or ETB evoked the increases in intracellular Ca2+ concentration ([Ca2+]i). ETB-mediated but not ET-1-induced [Ca2+]i increase was pertussis toxin (PTX) sensitive. Adenosine 3',5'-cyclic monophosphate (cAMP) formation via ETA was observed in PTX-treated cells, whereas the inhibition of isoproterenol-stimulated cAMP formation via ETB was observed in PTX-untreated cells. Like the ETB-selective antagonists, PTX treatment or dibutyryl cAMP partially (50-70%) inhibited ET-1-induced DNA synthesis. These data suggest that 1) ET-1 induces DNA synthesis predominantly through ETA, via PTX-insensitive G protein; 2) ETA-mediated cAMP formation inhibits DNA synthesis; and 3) stimulation of ETB coupling to Gi protein modulates ETA-mediated DNA synthesis by inhibiting cAMP formation.

Cell Division↗
PubMed1995

Protective effect of disaccharides on restriction endonucleases during drying under vacuum.

Desiccation by vacuum-drying inactivates the restriction endonuclease HindIII completely. However, when dried in the presence of a disaccharide such as trehalose, maltose, or sucrose, the endonuclease retains its lambda DNA-cleaving activity and produces the same digestive fragments as does the intact enzyme. Thus, the disaccharides are effective in protecting the restriction enzyme in terms of both recognition and accurate cleavage of the substrate. Among the disaccharides, trehalose protects the enzyme most effectively; and it also stabilizes the enzyme during dilution in aqueous solution. The restriction enzyme dried with trehalose maintains its activity without detectable loss for at least 4 days at 37 degrees C, but it shows reduced activity after 30-day storage at either 4 degrees C or room temperature. Trehalose also protects other restriction endonucleases, EcoRI and BamHI, from inactivation during vacuum-drying, whereas drying them alone leads to severe loss of their activity. The restriction endonucleases dried with trehalose retain their activities for at least 20 days at 4 degrees C and for 7 days at room temperature.

DNA Restriction Enzymes↗
PubMed1995

Solution structure of a human calcitonin analog elucidated by NMR and distance geometry calculations.

Three-dimensional structure of a human calcitonin analog (abbreviated as hCTa) in which the amino acids of the wild type are replaced at position 12, 16 and 19 by leucine residues and further at position 22 by a tyrosine residue was studied in TFE solution by 1H-NMR and distance geometry calculations. This analog has a 15-20 times activity as compared with the wild type. The amino acid replacements resulted in formation of an amphiphilic alpha-helix in the region between the residues 4-20. The overall three-dimensional structure is similar to that of the wild type. The conformational feature of hCTa with a hydrophobic face composed with a Met and four Leu residues may be related to its higher hypocalcemic potency.

Amino Acid Sequence↗
PubMed1995

In vivo administration of IL-1 beta accelerates silk ligature-induced alveolar bone resorption in rats.

The effects of recombinant human interleukin-1 beta (rhIL-1 beta) on alveolar bone resorptive activity in rats were examined. Continuous administration of rhIL-1 beta or phosphate-buffered saline (PBS) was given via osmotic pumps for 3, 7 and 14 days to rats with silk ligatures around second maxillary molars. Other animals without ligatures received insertion of pumps containing rhIL-1 beta or remained untreated. Sections were subject to three different stains:--hematoxylin and eosin (H-E) for histology, acid phosphatase (ACPase) activity for osteoclast detection, and immunohistochemistry using anti-rat monocyte/macrophage monoclonal antibody (ED 1). In addition, body weight, plasma calcium and phosphorus levels were monitored. The mean body weight of rats receiving rhIL-1 beta was significantly lower (P < 0.05 to P < 0.01) compared with untreated rats throughout the experimental period. On Day 7, plasma calcium and phosphorus levels were significantly lower in rats receiving rhIL-1 beta than in rats receiving PBS only (P < 0.05). Sections revealed a moderate inflammatory cell infiltrate reaching near the alveolar crest in both groups with ligatures on Day 3. Only rats receiving rhIL-1 beta exhibited enhancement of inflammatory cell invasion on Days 7 and 14. In rats receiving rhIL-1 beta with ligatures, numerous resorption lacunae containing ACPase-positive multinucleated giant cells (MNGCs), coinciding with ED1-positive cells, were located on the mesial side of the septum where extensive bone resorption had occurred throughout the experimental period. In animals receiving rhIL-1 beta without ligatures, compared with untreated rats, increased ACPase-positive cells were observed on the mesial side of the septum on Day 3. In animals receiving PBS only, a few ACPase-positive cells were observed confined to the mesial regions where slight bone resorption occurred on Days 7 and 14. These results indicate that the administration of rhIL-1 beta accelerated alveolar bone destruction in ligature-induced periodontal tissue inflammation over a two-week period.

Acid Phosphatase↗
PubMed1995

Indication for splenectomy combined with total gastrectomy for gastric cancer: analysis of data on 249 gastric cancer patients.

This study analyzed intraoperative indication for splenectomy at the time of total gastrectomy, based on 249 gastric cancer patients. Data on these patients were studied with special reference to the relationship between intraoperative gross findings such as serosal invasion, tumor size, histologic patterns and nodal metastasis to the splenic hilus. Fifty-three of the 249 patients (21.3%) had a positive metastasis. The incidence of nodal metastasis to the splenic hilus was high in patients with a primary lesion in the entire stomach (26/72: 36.1%). In case of no serosal invasion of the primary lesion, there was no nodal involvement to the splenic hilus (0/42). The low incidence of nodal metastasis occurred in case of a tumor size of less than 40 mm in the largest diameter (1/60: 1.7%) and with histological findings of signet ring cell carcinoma (1/19: 5.3%). Although site and size of the primary tumor, depth of tumor penetration, and histologic findings are interdependent variables, these factors indicate probable nodal involvement to the splenic hilus and unnecessary splenectomy can be avoided.

Female↗