Biomedical subjects

K Sakamoto

Publications and source records attributed to K Sakamoto.

At least 235 records · Page 13Linked to original sources

PubMed1998

Humoral signals mediate the circadian expression of rat period homologue (rPer2) mRNA in peripheral tissues.

Northern blot analysis revealed the circadian expression of rat period homologue (rPer2) mRNA in peripheral mononuclear leukocytes that have no neuronal connections. The mRNA expression in the suprachiasmatic nucleus (SCN), peripheral mononuclear leukocytes, and other peripheral tissues exhibited prominent, synchronous circadian oscillation with peaks at early night. As we have previously shown the peripheral circadian expression of rPer2 mRNA is abolished in SCN-lesioned rats [Sakamoto, K., Nagase, T. Fukui, H., Horikawa, K., Okada, T., Tanaka, H., Sato, K., Miyake, Y., Ohara, O., Kako, K. and Ishida, N., J. Biol. Chem., 273 (1998) 27039-27042], our findings suggest that some humoral signals, driven by the SCN, mediate the circadian expression of mammalian per homologues in peripheral tissues.

Animals↗

PubMed1998

Role of the isoprenyl tail of ubiquinone in reaction with respiratory enzymes: studies with bovine heart mitochondrial complex I and Escherichia coli bo-type ubiquinol oxidase.

The hydrophobic isoprene tail of ubiquinone-2 (Q2) exihibits binding specificity in redox reactions with bovine heart mitochondrial complex I (Ohshima, M., Miyoshi, H., Sakamoto, K., Takegami, K., Iwata, J., Kuwabara, K., Iwamura, H., and Yagi, T. (1998) Biochemistry 37, 6436-6445) and the Escherichia coli bo-type ubiquinol oxidase (Sakamoto, K., Miyoshi, H., Takegami, K., Mogi, T., Anraku, Y., and Iwamura, H. (1996) J. Biol. Chem. 271, 29897-29902). To identify the structural factor(s) of the diprenyl tail of Q2 governing the specific interaction with these enzymes, we synthesized a series of novel Q2 analogues in which only one of the structural factors of the diprenyl tail was systematically modified. In bovine complex I, the presence of the methyl branch and the pi-electron system in the first isoprene unit are responsible for high-affinity binding of Q2 to the ubiquinone reduction site, which results in a low Km and kcat values of Q2 reduction. The position of the methyl group in the tail is strictly recognized by the enzyme. In contrast to complex I, in bo-type ubiquinol oxidase, either of the two pi-electron systems in the tail is required for high-affinity binding of Q2H2 to the enzyme, while the presence of the methyl branch and the location of the pi-electron systems are not strictly recognized by the enzyme. We concluded that the role of the ubiquinone tail is not simply the enhancement of the hydrophobicity of the molecule and that molecular recognition of the tail by the quinone redox site differs among the respiratory enzymes.

Animals↗

PubMed1998

Translocation of HSP27 to cytoskeleton by repetitive hypoxia-reoxygenation in the rat myoblast cell line, H9c2.

We investigated the possible changes in the distribution of HSP27 after a brief hypoxia-reoxygenation stress in the rat myoblast cell line, H9c2, as a model of ischemic preconditioning. Cells were exposed to 4 cycles of 5 min. of hypoxia and 5 min. of reoxygenation. In the normoxic condition, HSP27 was exclusively found in the cytosolic fraction. After the hypoxia-reoxygenation cycle, HSP27 redistributed to the cytoskeletal fraction, which was blocked by 10 microM SB 203580, a specific inhibitor of p38 MAP kinase. Cells treated with the repetitive hypoxia-reoxygenation developed resistance against cell death induced by hypoxia for 24 hours. The changes in localization of HSP27 found in the present study may reflect the mechanism of preconditioning in the cardiac myocyte.

Animals↗

PubMed1998

Multitissue circadian expression of rat period homolog (rPer2) mRNA is governed by the mammalian circadian clock, the suprachiasmatic nucleus in the brain.

The period (per) gene, controlling circadian rhythms in Drosophila, is expressed throughout the body in a circadian manner. A homolog of Drosophila per was isolated from rat and designated as rPer2. The rPER2 protein showed 39 and 95% amino acid identity with mPER1 and mPER2 (mouse homologs of per) proteins, respectively. A robust circadian fluctuation of rPer2 mRNA expression was discovered not only in the suprachiasmatic nucleus (SCN) of the hypothalamus but also in other tissues including eye, brain, heart, lung, spleen, liver, and kidney. Furthermore, the peripheral circadian expression of rPer2 mRNA was abolished in SCN-lesioned rats that showed behavioral arrhythmicity. These findings suggest that the multitissue circadian expression of rPer2 mRNA was governed by the mammalian brain clock SCN and also suggest that the rPer2 gene was involved in the circadian rhythm of locomotor behavior in mammals.

Amino Acid Sequence↗

PubMed1998

Clinical studies on three cases of the interval form of carbon monoxide poisoning: serial proton magnetic resonance spectroscopy as a prognostic predictor.

Three patients with the interval form of carbon monoxide (CO) poisoning were studied for associations between their clinical courses and serial changes on: (1) MRI; (2) EEG; (3) single photon emission computed tomography with N-isopropyl-p-[123I]iodoamphetamine ([123I]IMP SPECT); and (4) proton magnetic resonance spectroscopy ([1HIMRS) to evaluate their usefulness as prognostic predictors. A hyperintense area on MRI T2-weighted images and a hypointense area on T1-weighted images, appearing in the deep white matter, persisted for a prolonged period even after improvement of the clinical symptoms, and did not become an accurate indicator of clinical evaluation or prognosis. [1H]MRS studies with the volume of interest set in the frontal lobe white matter revealed increases in choline-containing compounds, and reductions of N-acetylaspartate in all cases. These findings normalized in one subclinical case over time. Distinctive findings in the severe cases included increased lactate in two cases showing akinetic mutism, with a difference in prognosis noted according to the degree and period of persistence of this increase. EEG findings were relatively well correlated with clinical symptoms in the early period, with a good correlation no longer consistent after a certain period was exceeded. [123I]IMP SPECT findings did not always reflectclinical symptomatology either. These results indicate that [1H]MRS is the most useful indicator in the clinical evaluation of patients with the interval form of CO poisoning.

Adult↗

PubMed1998

Isolation and characterizations of quinone analogue-resistant mutants of bo-type ubiquinol oxidase from Escherichia coli.

Cytochrome bo is a member of the heme-copper terminal oxidase superfamily and serves as a four-subunit ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli. To probe the location and structural properties of the ubiquinol oxidation site, we isolated and characterized five or 10 spontaneous mutants resistant to either 2,6-dimethyl-1,4-benzoquinone, 2,6-dichloro-4-nitrophenol, or 2,6-dichloro-4-dicyanovinylphenol, the potent competitive inhibitors for the oxidation of ubiquinol-1 [Sato-Watanabe, M., Mogi, T., Miyoshi, H., Iwamura, H., Matsushita, K., Adachi, O., and Anraku, Y. (1994) J. Biol. Chem. 269, 28899-28907]. Analyses of the growth yields and the ubiquinol-1 oxidase activities of the mutant membranes showed that the mutations increased the degree of the resistance to the selecting compounds. Notably, several mutants showed the cross-resistance. These data indicate that the binding sites for substrate and the competitive inhibitors are partially overlapped in the ubiquinol oxidation site. All the mutations were linked to the expression vector, and 23 mutations examined were all present in the C-terminal hydrophilic domain (Pro96-His315) of subunit II. Sequencing analysis revealed that seven mutations examined are localized near both ends of the cupredoxin fold. Met248Ile, Ser258Asn, Phe281Ser, and His284Pro are present in a quinol oxidase-specific (Qox) domain and proximal to low-spin heme b in subunit I and the lost CuA site in subunit II, whereas Ile129Thr, Asn198Thr, and Gln233His are rather scattered in a three-dimensional structure and closer to transmembrane helices of subunit II. Our data suggest that the Qox domain and the CuA end of the cupredoxin fold provide the quinol oxidation site and are involved in electron transfer to the metal centers in subunit I.

Amino Acid Sequence↗

PubMed1998

Molecular cloning of serotonin N-acetyltransferase gene from the mouse and its daily expression in the retina.

The primary structure of serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AA-NAT: the rate-limiting enzyme in melatonin synthesis) in the mouse retina was deduced from the cDNA nucleotide sequence. The deduced protein consisted of 205 amino-acid residues with sequences highly conserved in AA-NATs of vertebrates, and was 96% identical to rat AA-NAT. Northern blot analysis of mouse retinal mRNA showed two obvious bands, of 1.5 kb and 4.5 kb in length. The levels of both transcripts were low at day and high at night, but the night-to-day ratios were <2. These findings suggest that the expression mechanism of AA-NAT transcripts in the mouse retina may be different from those in other mammals, where a single transcript of AA-NAT is normally observed in Northern blots.

Amino Acid Sequence↗

PubMed1998

Specificity of pyridinium inhibitors of the ubiquinone reduction sites in mitochondrial complex I.

Dual binding sites for pyridinium-type inhibitors in bovine heart mitochondrial complex I have been proposed (Gluck, M. R., Krueger, M. J., Ramsay, R. R., Sablin, S. O., Singer, T. P., and Nicklas, W. J. (1994) J. Biol. Chem. 269, 3167-3174). The marked biphasic nature of the dose-response curve for inhibition of the enzyme by MP-6(N-methyl-4-[2-(p-tert-butylbenzyl)propyl]pyridinium) makes this compound the first selective inhibitor of the two sites (Miyoshi, H., Inoue, M., Okamoto, S., Ohshima, M., Sakamoto, K., and Iwamura, H. (1997) J. Biol. Chem. 272, 16176-16183). Modifications of the structure of MP-6 show that a tert-butyl group on the benzene ring, a methyl group attached to the pyridine nitrogen atom, para-substitution pattern in the pyridine ring, and the presence of a branched structure in the spacer moiety are important for the selective inhibition. On the basis of the structural specificity, we synthesized a selective inhibitor, MP-24 (N-methyl-4-[2-methyl-2-(p-tert-butylbenzyl)propyl]pyridinium), which elicits greater selectivity. Characterization of the inhibitory behavior of MP-24 provided further strong evidence for the dual binding sites model.

Animals↗

PubMed1998

The localization of the site of arylalkylamine N-acetyltransferase circadian expression in the photoreceptor cells of mammalian retina.

To investigate the molecular mechanism of the melatonin rhythm in the mammalian retina, we examined the temporal mRNA expression pattern of arylalkylamine (serotonin) N-acetyltransferase (AA-NAT), the rate-limiting enzyme in melatonin synthesis in the rat retina. Rat AA-NAT mRNA was detected exclusively in the retinal photoreceptors in the outer nuclear layer--low during the day and increased more than threefold at night. The nocturnal AA-NAT expression in rat retina was also confirmed by RNase protection and the AA-NAT enzymatic activity. This is the first report to localize the site of AA-NAT mRNA circadian expression in mammalian photoreceptor cells.

Animals↗

PubMed1998

Characterization of the ubiquinone reduction site of mitochondrial complex I using bulky synthetic ubiquinones.

A wide variety of alkyl derivatives of Q2 (6-geranyl-2, 3-dimethoxy-5-methyl-1,4-benzoquinone) and DB (6-n-decyl-2, 3-dimethoxy-5-methyl-1,4-benzoquinone), in which methoxy groups of the 2- and/or 3-positions of the quinone ring were replaced by other bulky alkoxy groups from ethoxy to butoxy, were prepared by novel synthetic procedures. Electron-accepting activities of the bulky quinones were investigated with bovine heart mitochondrial complex I and its counterpart of Paracoccus denitrificans(NDH-1) to elucidate structural and functional features of the quinone reduction site of the enzymes. The bulky quinone analogues served as sufficient electron acceptors from the physiological quinone reduction site of bovine complex I. Considering the very poor activities of even the ethoxy derivatives as substrates for other respiratory enzymes such as mitochondrial complexes II and III [He, D. Y., Gu, L. Q., Yu, L., and Yu, C. A. (1994) Biochemistry 33, 880-884], this result indicated that the quinone reduction site of bovine complex I is spacious enough to accommodate bulky exogenous substrates. In contrast to bovine complex I, bulky quinone analogues served as poor electron acceptors with Paracoccus NDH-1. These observations indicated that bovine complex I recognizes the substrate structure with poor specificity. The substituent effects in the 2- and 3-positions of the quinone ring on the electron-transfer activity with bovine complex I differed significantly between Q2 and DB series despite having the same total number of carbon atoms in the side chain. The inhibitory effect involving Q2 due to its geranyl side chain was markedly diminished by structural modifications of the quinone ring moiety. These findings indicate that the side chain plays a specific role in the redox reaction and that the quinone ring and side-chain moieties contribute interdependently to binding interaction. Moreover, structural dependency of the proton-pumping activity of the quinone analogues was comparable to that of the electron-transfer activity with bovine complex I, indicating that the mechanism of redox-driven proton-pumping does not differ depending upon the substrate structure.

Animals↗

PubMed1998

5-hydroxydecanoate selectively reduces the initial increase in extracellular K+ in ischemic guinea-pig heart.

The aim of the present study was to determine the effect of 5-hydroxydecanoate (5-HD) on extracellular K+ levels during global ischemia for 30 min employing K+-sensitive electrodes in isolated guinea-pig hearts. 5-HD (100 microM) reduced the K+ accumulation during the early period of ischemia, but did not inhibit the elevation of extracellular K+ in the latter half of the ischemic period which was selectively enhanced by ouabain (3 microM). Thus, 5-HD appears to exert a similar mode of action as glibenclamide on extracellular K+ accumulation in the ischemic guinea-pig hearts. The present study also strengthens the previous conclusion that an ATP-sensitive K+ channel contributes only to the initial increasing phase of extracellular K+ accumulation during ischemia in guinea-pig hearts.

Animals↗

PubMed1998

Ectopic expression of lunatic Fringe leads to downregulation of Serrate-1 in the developing chick neural tube; analysis using in ovo electroporation transfection technique.

Lunatic Fringe (l-Fng) is one of the vertebrate homologues of Drosophila Fringe, which interacts with the Notch signal pathway and regulates activation of the Notch ligands, Delta and Serrate. To elucidate the roles of l-Fng in vertebrate neurogenesis, we transfected chick l-Fng (C-l-Fng) to chick neural tube using the in ovo electroporation technique and examined the subsequent changes in expression of Notch-related genes. We observed downregulation of C-Serrate-1 by ectopic C-l-Fng expression which implied that C-l-Fng acts on the vertebrate Notch pathway to regulate the expression of its ligand.

Animals↗

PubMed1998

Circadian expression of serotonin N-acetyltransferase mRNA in the rat retina.

To investigate the molecular mechanism of the melatonin rhythm in the mammalian retina, we examined the temporal mRNA expression pattern of serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AA-NAT; the rate-limiting enzyme in melatonin synthesis) in the rat retina. Northern blot analysis showed that in a daily light-dark cycle retinal AA-NAT mRNA was low during the day and increased more than threefold at night, and this daily rhythm persisted even in constant darkness. These findings suggest that AA-NAT mRNA expression in the rat retina is regulated by an endogenous circadian clock.

Animals↗

PubMed1998

An RNA aptamer to the xanthine/guanine base with a distinctive mode of purine recognition.

RNAs that bind to xanthine (2,6-dioxypurine) were isolated from a population of 10(12) random sequences by in vitro selection. These xanthine-binding RNAs were found to have a 10 nt consensus sequence at an internal loop in the most probable secondary structure. By trimming one of the xanthine-binding RNAs, a representative xanthine-binding RNA (designated as XBA) of 32 nt residues was prepared. The dissociation constant of this RNA for xanthine was determined to be 3.3 microM by equilibrium filtration experiments. The XBA RNA can bind to guanine as well, whereas it hardly accommodates adenine, cytosine or uracil. The K d values for various xanthine/guanine analogues were determined, and revealed that the N1H, N7 and O6 moieties of the ligand are involved in the binding with the XBA RNA. The ribonuclease sensitivities of some internal-loop residues changed upon the addition of xanthine, suggesting that the internal loop of the XBA RNA is involved in the ligand binding. Interestingly, the consensus sequence of the xanthine/guanine-binding RNAs is the same as a sequence in one of the internal loops of the hairpin ribozyme, except for a substitution that is neutral with respect to xanthine/guanine binding.

Base Sequence↗

PubMed1998

Effect of environmental factors on the dominant lethality caused by expression of a mutated DnaA protein with decreased intrinsic ATPase activity.

Induction of a mutant DnaA protein (DnaA E204Q) with decreased intrinsic ATPase activity in cells was previously shown to cause overinitiation of chromosomal DNA replication and a dominant lethal phenotype. Here it is shown that the dominant lethality required incubation at high temperatures; cells harboring the expression plasmid of DnaA E204Q showed very weak colony formation ability (less than 1/10(5) that of the wild-type DnaA) at 42 degrees C, whereas they showed a normal colony formation ability at 28 degrees C on LB agar plates. Overinitiation of chromosomal DNA replication caused by expression of DnaA E204Q also required incubation at high temperatures in LB medium. When the incubation was performed in synthetic (Tanaka) medium at 42 degrees C, neither the dominant lethality nor overinitiation caused by expressing DnaA E204Q was observed. These results suggest that the dominant lethality and the overinitiation caused by expressing DnaA E204Q require culture conditions that provide a high potential for cell growth.

Adenosine Triphosphatases↗

PubMed1998

Comparisons on chemically-induced mutation among four bacterial strains, Salmonella typhimurium TA102 and TA2638, and Escherichia coli WP2/pKM101 and WP2 uvrA/pKM101: collaborative study II.

A collaborative study of chemically-induced mutation was performed using the four bacterial strains Salmonella typhimurium TA102 and TA2638, and Escherichia coli WP2/pKM101 and WP2 uvrA/pKM101, used in the previous successive studies in order to compare the specific spectrum of response to chemicals among the four strains and to determine the usefulness (sensitivity) of each strain. Twenty-two laboratories participated in this study. Following the previous study, 28 compounds were selected consisting of oxidative agents or crosslinking agents. These compounds were tested for mutagenicity using the plate incorporation method with or without metabolic activation. The tests were performed in two laboratories per chemical. The number of chemicals which showed positive results were 18, 17, 16 and 14 with WP2 uvrA/pKM101, TA2638, TA102 and WP2/pKM101, respectively. In all four strains 19/28 chemicals (68%) were either positive or negative while the remaining 9 chemicals showed a heterogeneous response. With TA102 and TA2638 89% (25/28) of the test chemicals showed the same response, while 75-79% (21-22/28) showed the same response in these two S. typhimurium strains and in WP2 uvrA/pKM101. In conclusion, WP2/pKM101 strain showed the least sensitivity to the chemicals used. Among the other three strains, WP2 uvrA/pKM101 was the most sensitive strain, although the difference in the number of chemicals which showed positive results was minor. Concerning the spectrum of response to chemicals, it is apparent that TA102 and TA2638 have almost the same sensitivity, but some chemicals induce different responses between the two S. typhimurium strains and WP2 uvrA/pKM101.

Adenosine Triphosphatases↗

PubMed1998

Sensitization of cells to ionizing radiation by chlorin e6Na.

We have investigated whether a hydrophilic photosensitizer, chlorin e6Na (Ce6Na), can sensitize cells to ionizing radiation. When V79-1 cells were pretreated with Ce6Na for 2 hr before receiving a dose of 2-14 Gy of irradiation, the cells became sensitive to X-irradiation. The sensitizing effect of Ce6Na depended on the dose of Ce6Na. The sensitizing effect also depended on the length of the treatment period before X-irradiation up to 4 hr, but not on the length of a treatment period after X-irradiation. Intracellular concentrations of Ce6Na were increased linearly after incubation with Ce6Na for periods of up to 4 h. The dose-modifying factor calculated from the survival curve was 1.25.

Animals↗

PubMed1998

Differential adaptations of insulin-like growth factor-I, basic fibroblast growth factor, and leukemia inhibitory factor in the plantaris muscle of rats by mechanical overloading: an immunohistochemical study.

We investigated changes in several growth factors in the rat plantaris muscle produced by mechanical overloading by ablation of synergists using immunohistochemistry. At 1 and 3 days post surgery, the insulin-like growth factor-I (IGF-I) level was slightly increased in the cytosol and markedly increased in the invading cells of the extracellular space. Thereafter, the IGF-I immunoreactivity evoked by overloading rapidly decreased to the normal level. The level of leukemia inhibitory factor (LIF), which was not shown to change at 1 day post surgery, was increased in the cytosol at 3, 5, 7 and 10 days and at 2 weeks. Basic fibroblast growth factor (bFGF) immunoreactivity did not change during the entire period of overloading (1 day-3 weeks post surgery). These results indicate that the elevations of the levels of IGF-I and LIF show differential time course in the plantaris muscle subjected to functional overload. Furthermore, bFGF appears not to be related to the compensatory hypertrophy produced by overloading.

Adaptation, Physiological↗