Takamasa Hanaichi

Structural study of immunoaffinity-purified DNA polymerase α-DNA primase complex from calf thymus

The DNA polymerase alpha-DNA primase complex was purified over 17,000-fold to near homogeneity from calf thymus using an immunoaffinity column. Sodium dodecyl sulfate gel electrophoresis revealed three polypeptides with molecular weights of 140, 50 and 47 kDa, in a ratio of 1:2:0.25. The complex showed a sedimentation coefficient of 9.7 S, a Stokes radius of 56 A and a native molecular weight of 250-260 kDa. Taken together, the data suggest that the calf thymus dNA polymerase alpha-DNA primase complex is essentially a heterotrimer of large (140 kDa) and small (50 kDa) subunits in a ratio of 1:2, with a globular conformation. Electron-microscopic studies of the complex revealed a spherical particle of 120 A in diameter, in agreement with the physiochemical results. The binding of the complex to DNA was also demonstrated.

Gamete development in Plasmodium berghei regulated by ionic exchange mechanisms

Ionic regulation in the induction of exflagellation ofPlasmodium berghei was investigated by culturing the parasites in various isotonic media. Of the salts tested, NaHCO3 exhibited the highest activity in inducing exflagellation, whereas KHCO3 showed no activity. In the absence of HCO3−, media containing monovalent cation (Na+, K+, Cs+, Rd+, choline+, lysine+, arginine+) and Cl− also induced exflagellation, but their activities were lower than that of NaHCO3. Anions of Br− or NO3− could be substituted with Cl−, whereas other anions such as I−, NO2−, SO42−, SCN−, H2PO4−, or HPO42− failed to induce exflagellation, as did tetramethylammonium-Cl, CaCl2, MgSO4, MgCl2 and sucrose as well. These results suggest that the induction of exflagellation requires the presence of Na+ and HCO3− or monovalent, membrane-permeable cation and Cl− in the medium. Measurements of the efflux of H[14C]O3− or Cl− indicated that these anions were released from the cells into the NaCl or the NaHCO3 medium, respectively, probably by exchange in HCO3−/Cl−. Determination of intracellular ionic concentrations by electron microscopic X-ray microanalysis of cryopreserved specimens revealed that in the NaHCO3 medium, external Na+ (and probably HCO3−) enters the gametocytes by exchange with internal Cl− (and probably H+), whereas in Cl−-containing media, external unspecified cation and Cl− influx by exchange, probably with H+ and HCO3−. It is therefore suggested that two separate ion exchangers, i.e., Na+-dependent HCO3−(in)/Cl−(out) and nonspecific monovalent-cation-dependent Cl−(in)/HCO3−(out) exchangers, are involved in the induction of gametogenesis inP. berghei. Furthermore, the presence of both classes of anion in the medium enhanced exflagellation activity and increased Na+ uptake more than did the NaCl or NaHCO3 medium alone. The apparent synergistic enhancement by two contraactive anion exchangers is consistent with a “recycling” model of pHi regulation, in which HCO3− and Cl− are exchanged between the cells and the media, resulting in the acceleration of monovalent cation/H+ exchange.

Effects of dantrolene and methylxanthines on the sensory nerve terminal of the frog muscle spindle

The application of 1.5-4 microM dantrolene decreased the threshold and the current sensitivity of the rhythmic hyperpolarizations that occur during depolarization of the sensory nerve terminal in the frog muscle spindle. The higher concentration provoked spontaneous rhythmic changes even without depolarization. Methylxanthines (5 mM caffeine, theophylline or pentylene-tetrazole) increased the threshold and the sensitivity. Electron microscopic observations of the dantrolene-treated spindles revealed numerous electron-dense deposits associated with the cytoplasmic membrane of the sensory terminals and with mitochondrial membranes. The deposits were found to contain K+ and Ca2+ by energy dispersive X-ray microanalysis. Electron-dense deposits containing Ca2+ were usually observed in the inner capsular space and in the mitochondria of the sensory terminals perfused by normal or high Ca2+ Ringer solutions. They were reduced in number following incubation with methylxanthines. The amplitudes of afferent spikes and the spindle potential were increased by methylxanthines in much the same way as by K+ channel blockers, suggesting that GK of the terminal membrane may be reduced by methylxanthines. We suggest that methylxanthines may modulate the terminal responses both as a K+ channel blocker and by enhancing the release of Ca2+ from a storage site, perhaps in the inner capsular space, whereas dantrolene has the opposite effect.

Acceleration of Deactivation of Ag Catalyst by Periodic Pulse Technique for Rapid Estimation of Catalyst Life

Abstract In order to examine the possibility of the rapid estimation of catalyst life by a periodic pulse technique, the deactivation of Ag catalyst in the oxidation of C2H4 was examined. In the periodic pulse technique, O2+N2 mixture and C2H4+N2 mixture were fed alternately to subject a catalyst to forced redox cycle, which is expected to accelerate the sintering of catalyst under the reaction condition. As expected, the deactivation was faster in the periodic pulse technique than in the continuous flow technique. TEM also indicated that the particle size of Ag increased faster in the periodic pulse run. Although the deactivation was the fastest in O2+N2 environment, the deactivation behavior was different from that under the reaction condition. It was shown that the deactivation should be accelerated under the reaction condition for the rapid estimation of catalyst life, and that the periodic pulse technique is useful for such purpose.