[Primary structure and function of proton-translocating ATPase of Escherichia coli].
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Biomedical subjects
Publications and source records attributed to H Kanazawa.
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A radiolucent silicone spacer was developed for use in interstitial irradiation for cancer of the tongue. It keeps about eight millimeters of distance between the lower gingiva and the lesion of the tongue, and the average radiation dose (TDF) to the lower gingiva was 45% of the minimal tumor dose in the lingular lesion. The total TDF value of the lower gingiva by both external and interstitial irradiation was between 66 and 114, which is not considered to be responsible for the occurrence of osteoradionecrosis of the mandible.
A comparative study of the serum, atrial muscle, and pericardial fluid concentrations of latamoxef (LMOX) and cephalothin (CET) was performed in 18 adult patients having cardiac surgery. Patients were randomly assigned to receive either a single dose of CET 30 mg/kg, LMOX 30 mg/kg, or LMOX 20 mg/kg. Each drug was administered intravenously soon after anesthetic maintenance was achieved. After each drug administration, serum samples were obtained at various time intervals before, during, and after extracorporeal circulation (ECC), and assayed for CET and LMOX concentration. A sample of pericardial fluid was collected immediately after the pericardium was opened. The right atrial appendage tissue samples were obtained at the time of heart cannulation. The pharmacokinetics and penetration characteristics of each drug were analysed by the method of two-compartment model. Serum concentrations of CET following a single intravenous injection of 30 mg/kg were the high values, 172.4 +/- 35.6 micrograms/ml at 5 minutes, and 92.9 +/- 33.8 micrograms/ml at 15 minutes after injection. The initial (before bypass) half-life was 0.22 +/- 0.11 hour (13.1 minutes) while half-lives during and after ECC were prolonged significantly. In the cases of CET group, the myocardial tissue concentration value of 4.86 micrograms/g was obtained in only 1 case. The mean pericardial fluid concentration was 4.69 +/- 3.35 micrograms/ml at 5 to 30 minutes after injection. Serum concentration of LMOX following a injection of 30 mg/kg were 253.6 +/- 67.4 micrograms/ml at 5 minutes, and 176.3 +/- 64.1 micrograms/ml at 15 minutes after injection.(ABSTRACT TRUNCATED AT 250 WORDS)
From 1965 to 1982, 195 cases of maxillary cancer receiving more than 30 Gy of radiotherapy and surviving more than 6 months were investigated. Chronic cerebral necrosis following irradiation was found in 5 cases. All of them were irradiated with a total dose of more than 60 Gy. Symptoms caused by radiation injury of the brain were observed after more than 3 years. CT images showed irregular low-density areas in the base of the frontal or the temporal lobe within the irradiated field, and the lesion was enhanced in 16.6% of the patients who were irradiated with more than 60 Gy and survived without local recurrence for more than 3 years. Their symptoms, pathology, treatment, prognosis and procedures to avoid chronic cerebral damage were also discussed.
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To investigate the weight-bearing function of the fibula, weight-loading experiments were performed on autopsy specimens. With the ankle joint in neutral position, the weight distribution to the fibula amounted to 6.4%. With dorsiflexion of the ankle joint, the weight on the fibula increased. With plantar flexion of the ankle joint, the weight on the fibula decreased. Lateral and posterior loading of the tibia produced increased weight on the fibula. With eversion of the ankle joint, the weight on the fibula increased. The weight-bearing function increased with augmentation of the tibiofibular articular angle. These characteristics of the fibular weight-bearing function should be considered carefully in the treatment of the knee joint or the fibula.
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Complete nucleotide sequence of the genes for subunits of the H+ ATPase of E.coli has been determined and several hybrid plasmids carrying various portions of these genes have been constructed. Genetic complementation and recombination tests of about forty mutants of E.coli defective in the ATPase were performed using these plasmids for identifying the locations of the mutations. Two mutants defective in the delta subunit and a novel type of mutant defective in the b subunit of F0 were identified. The delta subunit mutants showed no proton conduction, suggesting that this subunit has an important role for the proton conduction. The ATPase of the b subunit mutant has a normal activity of proton channel portion, which phenotype is clearly different from that of mutants of the b subunit reported previously.
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Conformational change in the alpha subunit of Escherichia coli proton-translocating ATPase was studied using trypsin. The subunit was cleaved with a small amount of trypsin (1 microgram/mg subunit) to peptides of less than 8000 daltons. On the other hand, the subunit was cleaved to two main polypeptides (30,000 and 25,000 daltons) in the presence of sufficient ATP (1 mM-0.5 microM) to saturate the high-affinity site of the subunit. Analysis of digests of the subunit combined with fluorescent maleimide suggested that the subunit was digested in the middle of the polypeptide chain in the presence of the nucleotide. ADP and adenylyl imidodiphosphate had the same effect as ATP. These results suggest that the conformation of the subunit changed to form two trypsin-resistant domains upon binding of ATP to the high-affinity site.
Sixteen mutants of Escherichia coli defective in H+-ATPase (proton-translocating ATPase) were tested for their ability to recombine with hybrid plasmids carrying various portions of the beta subunit cistron. Twelve mutations were mapped within the carboxyl half of the cistron corresponding to amino acid residues 279 to 459 (domain II), while four mutations were mapped within residues 17 to 278 (domain I). The biochemical properties of these mutants were analyzed in terms of the proton permeability of their membranes and the assembly properties of their F1F0 complex. The mutants were classified according to the properties into three types, I, II, and III. In 12 mutants of type I, proton conduction in membrane vesicles was blocked and little F1 was released from the membranes under conditions in which F1 could be released from wild-type membranes, suggesting that assembly of the F1F0 complex is structurally and functionally defective. F1 was partially purified with very low recovery from one of the type I mutants, KF16. ATPase activity was reconstituted from this F1 with the beta subunit of the wild type, confirming the genetic results. Only one mutant, KF38, was classified as type II. Its membranes were partially leaky to protons and its F1 was releasable, suggesting that the interaction of its F1 and F0 was unstable. Type III mutants, KF11 and KF43, had an F1F0 complex with very low activity, in which the structure of F1 was relatively similar to that of the wild type. F1 was purified as a single complex from KF43 in this study and from KF11 previously (H. Kanazawa, Y. Horiuchi, M. Takagi, Y. Ishino, and M. Futai (1980) J. Biochem. 88, 695-703). Reconstitution experiments in vitro showed that the F1's of both mutants were defective in the beta subunit. The properties of the altered F1 of KF43 differed from those of F1 of KF11, suggesting that the mutation sites of KF43 and KF11 were different. From the results of mapping mutation sites and the biochemical properties of the mutants, the correlation of structural domains with function of the beta subunit is discussed. Most type I and type II mutations except that of KF39 were mapped in domain II, while the type III mutations were mapped in domain I, suggesting that domain II is more important than domain I for the function of the beta subunit, especially in terms of proper assembly of the F1F0 complex.
Various hybrid plasmids carrying a portion of the gene for the gamma subunit of the H+-ATPase of Escherichia coli complemented five mutants defective in the enzyme in a genetic test, indicating that the mutants are defective in the gamma subunit. Since the nucleotide sequence of genomic DNA carried on the plasmids is known, the defective site(s) of the mutants could be located within the gene for the gamma subunit as follows: KF10 and NR70, KF1, and KF12 and KF13 have a mutation causing a defect(s) in amino acid residues 1 to 82, 83 to 167, and 168 to 287, respectively, of the gamma subunit. The biochemical properties of all these mutants except NR70 were analyzed in terms of proton permeability of the membranes and assembly of F1. Results suggested that KF1 and KF10 have defective F1 without at least the alpha and beta subunits on their membranes, whereas KF12 and KF13 have F1's of rather similar structure to that of the wild type. Attempts were made to purify F1 oF KF12 as a single complex. Although the F1 complex dissociated during purification, active alpha and beta subunits of KF12 were partially purified. On the basis of these biochemical and genetic results, it is suggested that structural alterations in the primary sequence of the gamma subunit corresponding to residues 1 to 167 cause more extensive defects in the assembly of F1 than alteration in the sequence of residues 168 to 287.
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The combined effects of ACNU and X-irradiation on cultured HeLaS3 cells were investigated. Pretreatment with either ACNU or X-ray induced a substantial reduction in shoulder width the D0 value of the dose-response curve for the other agent, given later was unchanged. ACNU did not inhibit the recovery of sublethal damage (SLD) induced by X-ray when this treatment preceded the split-dose experiment. Our results indicate that some cell damage induced by each agent is transmissible to the progeny of the surviving cells and that the interaction of ACNU and X-irradiation was lethal to the cells.