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Y Kagawa

Publications and source records attributed to Y Kagawa.

At least 163 records · Page 9Linked to original sources
PubMed1990

Small-angle neutron scattering from the reconstituted TF1 of H(+)-ATPase from thermophilic bacterium PS3 with deuterated subunits.

Subunits alpha, beta and gamma of adenosine triphosphatase (H(+)-ATPase) from the thermophilic bacterium PS3 (TF1) have been over-expressed in Escherichia coli. alpha and beta subunits deuterated to the level of 90% were obtained by culturing E. coli in 2H2O medium. Both the subunits and the reconstituted alpha beta gamma complex, TF1, which contain the deuterated components in various combinations, were studied in solution by small-angle neutron scattering. The individual shapes of the subunits and their organization in the alpha beta gamma-TF1 complex were examined using the techniques of selective deuteration and contrast variation. The alpha and beta subunits are well approximated as ellipsoids of revolution having minor semi-axes of 20.4(+/- 0.4) and 20.0(+/- 0.2) A, and major semi-axes of 53.0(+/- 1.4) and 55.8(+/- 0.9) A, respectively. In the TF1 complex, three beta subunits are aligned to form an equilateral triangle, with their major axes tilted by 35 degrees with respect to the 3-fold axis of the complex. The beta-beta distance is about 53 A. Three alpha subunits are similarly arranged, positioned between the beta subunits, and with their direction of tilt opposite to that of the beta subunits. The centers of the alpha and beta subunits lie in the same plane, forming a hexagon. Adjacent subunits overlap in this model, suggesting that they are not simple ellipsoids of revolution.

Bacteria↗
PubMed1990

Novel regulatory enhancer in the nuclear gene of the human mitochondrial ATP synthase beta-subunit.

The system coordinating expressions of nuclear coded mitochondrial proteins was investigated by examination of the 5'-flanking region of the human mitochondrial ATP synthase beta-subunit gene. The promoter activity was measured by a transient expression of a chloramphenicol acetyltransferase (CAT) gene connected with various 5'-deletion mutants of the 5'-flanking region. In this experiment, at least two regions enhanced this promoter activity and at least one region repressed it. In one of the enhancing regions, a consensus sequence was found for the genes of other mitochondrial proteins such as those for cytochrome c1 (Suzuki, H., Hosokawa, Y., Nishikimi, M., and Ozawa, T. (1989) J. Biol. Chem. 264, 1368-1374) and the pyruvate dehydrogenase alpha-subunit (Maragos, C., Hutchison, W. M., Hayasaka, K., Brown, G. K., and Dahl, H.-H. M. (1989) J. Biol. Chem. 264, 12294-12298; Ohta, S., Endo, H., Matsuda, K., and Kagawa, Y. (1989) Ann. N. Y. Acad. Sci. 573, 458-460). The characteristics of this enhancing element were examined by introducing a synthetic oligonucleotide element into the CAT plasmid with a deleted enhancing element. The resulting plasmid showed full recovery of promoter activity, and this activity was independent of the orientation or location of the insert. Therefore, this is an enhancer that may be common to the nuclear genes of some mitochondrial proteins involved in energy transduction.

Base Sequence↗
PubMed1990

Marked decrease of mitochondrial DNA with multiple deletions in a patient with familial mitochondrial myopathy.

Muscle mitochondrial DNA (mtDNA) from a patient with mitochondrial myopathy was examined by Southern blotting. Her family history suggests autosomal dominant inheritance of this disorder. In contrast to other cases of this myopathy that are associated with a compensatory increase of mitochondria, in this patient, the content of mtDNA was markedly decreased (15% of controls), whereas protein of the ATP synthase beta-subunit was not decreased appreciably as judged by Western blotting. In addition, the mitochondrial DNA had multiple deletions, which were located between the replication origins of the heavy strand and light strand.

Adult↗
PubMed1990

Effects of ethidium bromide treatment of mouse cells on expression and assembly of nuclear-coded subunits of complexes involved in the oxidative phosphorylation.

Using mouse cell lines 5P and 5PEr (ethidium bromide-resistant derivative of 5P), we examined the influence of blocking expression of mitochondrial-gene products with ethidium bromide on the expression and assemblies of nuclear-coded subunits of the complexes involved in the oxidative phosphorylation. The results suggest that in the absence of mitochondrial-coded products, the expressions of subunit VIc of complex IV and beta-subunit of F1-ATPase are not affected, but that most nuclear-coded subunits other than alpha- and beta-subunits of F1-ATPase cannot be assembled nor inserted into the inner membrane.

Actins↗
PubMed1990

Regulation of mitochondrial ATP synthesis in mammalian cells by transcriptional control.

1. Mitochondrial ATP synthesis (oxidative phosphorylation) is mainly regulated by the membrane potential (respiratory control) and protein synthesis (transcriptional control). 2. According to the current view, genes for enzymes of oxidative phosphorylation are classified as housekeeping genes that are transcribed constitutively. These genes have been sequenced. 3. Four complexes of oxidative phosphorylation (complexes I, III and IV, and ATP synthase) are exceptional oligomers that contain subunits encoded by mitochondrial DNA. The remaining subunits of these complexes, as well as thousands of other mammalian enzymes are encoded by nuclear DNA. 4. It is proposed that ATP synthase (F0F1) and these oligomers supplying energy to F0F1, though they are housekeeping, are under some coordinated transcriptional control. Not transcription, but translation of mitochondrial DNA is mainly regulated. 5. Recently, studies on cloned human genes for the FoF1 beta subunit and 7 enzymes related to ATP synthesis revealed coordinated transcription. Moreover, a novel common cis-element (enhancer) was discovered in the 5'-upstream region of genes for the F1 beta subunit, cytochrome c1 and pyruvate dehydrogenase E1 alpha subunit. 6. In contrast to heme control and catabolite repression of yeast via trans-acting elements such as HAP and GP, signal transduction and coordinated transcription of human oxidative phosphorylation is not directed by CP1 (a HAP homolog), but may be closely related to cell differentiation.

Adenosine Triphosphate↗
PubMed1990

Partial amino acid sequence of an amyloid fibril protein from nodular primary cutaneous amyloidosis showing homology to lambda immunoglobulin light chain of variable subgroup III (a lambda III).

An amyloid fibril protein (MA) was purified as a 17,000-dalton protein from a case of nodular primary cutaneous amyloidosis, and its partial amino acid sequence (22 residues from N-terminal) was determined. A sequence closely homologous to that of the lambda III subgroup of the immunoglobulin light chain was detected. This is the third case of nodular primary cutaneous amyloidosis which has been studied at the level of sequence analysis of purified amyloid fibril proteins, and the first case of nodular primary cutaneous amyloidosis in which a lambda III amyloid protein has been shown to be present by sequence analysis.

Amino Acid Sequence↗
PubMed1990

Intra-arterial ACNU chemotherapy employing 20% mannitol osmotic blood-brain barrier disruption for malignant brain tumors.

The clinical effects and problems of intra-arterial water-soluble antitumor nitrosourea (ACNU) therapy following osmotic blood-brain barrier modification are discussed. Twenty-one patients with malignant brain tumors were divided into two groups. Group 1 consisted of 16 patients treated by operation, irradiation, and two or more courses of intracarotid infusion of ACNU 100 mg/body (1.7-2.2 mg/kg) following 20% mannitol 200 ml (1.3-1.6 ml/sec) (7 grade 4 astrocytomas, 5 grade 3 astrocytomas, and 4 others). Group 2 consisted of five patients treated by operation, irradiation, and repeated intracarotid infusion of ACNU 100 mg/body alone (grade 4 astrocytoma). The 2-year survival rate in Group 1 was 79% (11 of 14 cases followed up for longer than 2 years) and the 3-year survival rate was 67%. Five of seven grade 4 astrocytoma patients (71%) in Group 1 survived for more than 1 year 6 months, whereas four of five grade 4 astrocytoma in Group 2 died within 1 year 6 months. The measurement of the ACNU concentration in tumor tissues and blood in 11 brain tumors, after intracarotid infusion of ACNU with blood-brain barrier disruption, showed peak values in the tumor tissues of 3.02-32.53 micrograms/gm (mean, 9.67 micrograms/gm), about three to five times as high as that in blood in most cases. This method used in Group 1 appears to be relatively safe without permanent neurological deficits and offers a potential therapeutic effect when used in combination with appropriate premedication in suitable patients.

Adolescent↗
PubMed1989

Multiple deletions in mitochondrial DNA at direct repeats of non-D-loop regions in cases of familial mitochondrial myopathy.

Muscle mitochondrial DNAs from two brothers with mitochondrial myopathy associated with peripheral neuropathy had multiple deletions, most of which started in non-D-loop regions, unlike in an autosomal dominant mitochondrial myopathy (Zeviani, M. et al., Nature 339, 309 (1989)). The non-D-loop regions with deletions were amplified by the polymerase chain reaction and the resulting fragments were subcloned and then sequenced. At least 12 deletions of different lengths in different sites were found. However, all the deletions were flanked by short direct repeats (4-12 base pairs).

Base Sequence↗
PubMed1989

Deficiencies in complex I subunits of the respiratory chain in Parkinson's disease.

Immunoblotting studies on mitochondria prepared from the striata of patients who died of Parkinson's disease were performed using specific antisera against Complexes I, III and IV. In 4 out of 5 patients with Parkinson's disease, the 30-, 25- and 24-kDa subunits of Complex I were moderately to markedly decreased. No clear difference was noted in immunoblotting studies on subunits of Complexes III and IV between the control and Parkinson's disease. Deficiencies in Complex I subunits seem to be one of the most important clues to elucidate pathogenesis of Parkinson's disease.

Corpus Striatum↗
PubMed1989

Alpha 3 beta 3 complex of thermophilic ATP synthase. Catalysis without the gamma-subunit.

A complex of the alpha- and beta-subunits of thermophilic ATP synthase showed about 25% of the ATPase activity of the alpha beta gamma complex. The alpha 3 beta 3 hexamer structure was analyzed by sedimentation (11.2 S) and gel filtration (310 kDa). Dilution of the alpha beta complex caused dissociation of the complex and rapid loss of ATPase activity which was restored by addition of the gamma-subunit. A previous method using urea for isolating the subunits resulted in an alpha beta complex with lower activity than that prepared by over-expression of the genes. The alpha beta-ATP complex was formed from the alpha beta complex, ADP and Pi in the presence of dimethyl sulfoxide.

Bacteria↗
PubMed1989

Steady state kinetics of proton translocation catalyzed by thermophilic F0F1-ATPase reconstituted in planar bilayer membranes.

The proton-translocating ATPase of the thermophilic bacterium PS3 was reconstituted into planar phospholipid bilayers by the previously reported method (Hirata, H., Ohno, K., Sone, N., Kagawa, Y., and Hamamoto, T. (1986) J. Biol. Chem. 261, 9839-9843), and the relationship between the electric current induced by ATP and the concentration of ATP was examined. The magnitude of the electric current generated upon addition of ATP followed simple Michaelis-Menten type kinetics, and the Michaelis constant was found to be 0.14 mM under our conditions. This value is close to the values reported for F1- or F0F1-ATPase in its steady state catalytic cycle, indicating that the proton translocation is coupled to the steady state ATPase reaction. The relationship between the Km value and the membrane potential was also examined under the voltage-clamped condition, and we found that there was no apparent dependence of the Km on membrane voltage. These results together with the previous data suggest that the voltage dependence residues in some step that defines the apparent Vmax rather than Km in the reaction cycle, and proton translocation is not directly coupled to this ATP binding step.

Adenosine Triphosphate↗
PubMed1989

Cytochrome c oxidase--deficient myogenic cell lines in mitochondrial myopathy.

In a patient with mitochondrial myopathy, the defect of cytochrome c oxidase activity was restricted to some muscle fibers. To isolate cell lines with or without oxidase activity from a single muscle sample, primary cultured cells were transformed by replication origin-defective simian virus 40, and then cloned. The clones were examined by cytochemical staining for cytochrome c oxidase activity. Eight myogenic clones were completely devoid of activity, while the other myogenic and nonmyogenic clones were not. Deficiency of cytochrome c oxidase was stable in culture for at least a year after serial passaging. The amount of mitochondrial DNA in cytochrome c oxidase-deficient cells was the same as in control cells, and no deletion in the mitochondrial DNA was detected. Protein synthesis in mitochondria of the subunits of cytochrome c oxidase and subunit 6 of the ATP synthase complex was markedly decreased, whereas synthesis of the other subunits encoded by mitochondrial DNA was normal. These cloned cell lines provide an excellent system for clarifying the cause of mitochondrial myopathy and for investigating nuclear-mitochondrial genetic interaction.

Adolescent↗
PubMed1989

Synergistic interaction between etoposide and 1-beta-D-arabinofuranosylcytosine.

The sequence-dependency of the antitumor effect of etoposide (VP-16) and 1-beta-D-arabinofuranosylcytosine (ara-C) and its mechanisms were investigated in L1210 ascites tumor. Treatment with VP-16 (15 mg/kg) and ara-C (25 mg/kg) was administered intraperitoneally on days 1, 4, and 7 after tumor inoculation. Three-hour pretreatment with VP-16 followed by ara-C produced 70% of cure rate, but only 20% of cure rate was obtained with the reverse sequence. Simultaneous administration led to the worst therapeutic result. To clarify this sequence-dependent interaction of the both drugs, the effect of VP-16 on incorporation of ara-C into DNA was investigated in vivo. On day 3 after intraperitoneal transplantation of 1 x 10(6) L1210 cells, 15 mg/kg of VP-16 and 1 microCi of (3H)ara-C was administered intraperitoneally with or without time interval. The effect of VP-16 on ara-C incorporation was also strikingly schedule-dependent and compatible with the schedule dependency of the antitumor effect. At 1 hr after injection of ara-C, simultaneous administration of 15 mg/kg of VP-16 reduced ara-C incorporation by 67% of ara-C injection alone. But in sharp contrast to simultaneous-administration, 3 hour preadministration of VP-16 increased ara-C incorporation up to 247%. From 1 to 6 hour after intraperitoneal administration of 15 mg/kg of VP-16, the sedimentation rate of L1210 DNA was increasing on alkaline sucrose gradients. This repairing period from VP-16 induced DNA damage might be the vulnerable time to ara-C through an increase in ara-C incorporation into DNA.

Animals↗
PubMed1989

Hexagonal structure of two-dimensional crystals of the alpha 3 beta 3 complex thermophilic ATP synthase.

The highly dissociable alpha 3 beta 3 subunit complex (Mr = 319,582) of thermophilic ATP synthase was crystallized on a mercury surface under oxygen. The two-dimensional crystal was compared with that of TF1 (Mr = 385,351, alpha 3 beta 3 gamma delta epsilon subunit complex) by means of computer image processing. The crystals showed the same hexagonal lattice (a = b = 10 nm), despite the difference in their molecular weights. The color images of the two protein molecules were also hexagonal. However, there was an open hole in the image of the alpha 3 beta 3 complex, where small subunits (gamma, delta, and epsilon) of TF1 may have been located. The structure of this heterohexamer is consistent with that deduced from other physical parameters.

Crystallization↗