Microdosimetric measurements and estimation of human cell survival for heavy-ion beams

The microdosimetric spectra for high-energy beams of photons and proton, helium, carbon, neon, silicon and iron ions (LET = 0.5-880 keV/microm) were measured with a spherical-walled tissue-equivalent proportional counter at various depths in a plastic phantom. Survival curves for human tumor cells were also obtained under the same conditions. Then the survival curves were compared with those estimated by a microdosimetric model based on the spectra and the biological parameters for each cell line. The estimated alpha terms of the liner-quadratic model with a fixed beta value reproduced the experimental results for cell irradiation for ion beams with LETs of less than 450 keV/microm, except in the region near the distal peak.     

Approaches for submicrosequencing

From a conventional SDS-acrylamide gel plate, protein (1 g), even after staining by dye, was extracted with 70% formic acid and purified by Biogel P-10 column chromatography in 70% formic acid. The recovery was 60–99% and the protein purified by this method was free from SDS, dyes, glycine, and buffer salts and ready to use for protein composition and sequence studies. The method can avoid the protease digestion of the protein that has often been observed in the electroelution method. For amino acid analysis, protein was hydrolyzed with a gas of a HCL/trifluoroacetic acid/H₂O mixture at 158°C for 22.5 and 45 min to avoid contamination (less than 1 pmole). The method provided sufficient hydrolysis even for hydrophobic protein. It is under process of automation. Carboxyl-terminal analysis was performed with carboxypeptidases A, B, and P in the presence of alcohols and detergents. The effects on the carboxypeptidases (and several proteases) are summarized. These additions increased the solubility of proteins and enhanced the digestion. For N-terminal sequencing, conventional Edman degradation was followed up to the ATZ-derivative and this ATZ-derivative was reacted with primary amines such as ¹²⁵I-histamine or 1-aminopyrene to produce a phenylthiocarbamyl-amino acid-amine derivative with a yield as high as 95%. The conventional procedure of Edman degradation was preserved, but instead of the acid conversion to PTH derivatives, the above coupling procedure was introduced in order to increase the sensitivity to the 1–100f mole range.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Purification and Characterization of a Vegetative Lytic Enzyme Responsible for Liberation of Daughter Cells during the Proliferation ofChlamydomonas reinhardtii

A vegetative lytic enzyme (VLE) of Chlamydomonas reinhardtiimediates digestion of the cell walls of mother cells (sporangia)to allow release of daughter cells after mitotic cell divisionin the vegetative cell cycle. This enzyme is secreted into theculture medium concurrently with the appearance of daughtercells in synchronized cultures. Using an assay that monitorsdigestion of the mother cell wall, we purified VLE by ion-exchangeand gel-filtration chromatography from the medium of synchronizedcultures. The purified enzyme was a basic glycoprotein withan apparent molecular mass of 120 kDa on gel filtration and130 kDa on SDS-PAGE. Thus, VLE appeared to behave as a monomer.The enzyme acted specifically on the mother cell wall and wasunable to digest the cell walls derived from single vegetativecells. The enzymatic activity was inhibited by PMSF, p-APMSF,TLCK, HgCl₂, iodoacetate, EGTA, EDTA and 1,10-phenanthroline.VLE cleaved several synthetic model peptides on the carboxylside of a Lys or Arg residue, indicating that it is a proteasethat acts on protein in the mother cell wall in vivo to releasethe daughter cells. (Received November 30, 1994; Accepted March 22, 1995)     

Responses of single olfactory receptor cells to sex pheromones in the tobacco cutworm moth, Spodoptera litura

A single olfactory receptor cell of the male tobacco cutworm moth was recorded at the basal region of the sensillum trichodeum and its response to pheromones. Low frequency impulses were recorded from the olfactory receptor to compound A alone. Stimulation with a mixed ratio (9:1) of compound A and compound B increased the frequency of impulses more than compound A alone. However, increase of compound B in the ratio (5:5, 1:9) reduced gradually the frequency of impulses. Stimulation by compound B alone was without effect.It appears that the synergistic effect probably involve mechanisms at the level of the olfactory receptor cell.     

Dynamics of Ca(2+)/calmodulin-dependent protein kinase II following acute myocardial ischemia-translocation and autophosphorylation

Ca(2+)/calmodulin-dependent protein kinase (CaMK) family is responsive to changes in the intracellular Ca(2+) concentration. However, their functions have not been well established in the ischemia/reperfusion heart. The effects of myocardial ischemia on CaMKII, the most strongly expressed form, were investigated using isolated rat hearts. Rat hearts were rendered globally ischemic by stopping perfusion for 15 min, and then reperfused, heart ventricles being analyzed in each phase. Western blotting detected a decrease in the cytosolic and concomitant increase in the particulate fraction of CaMKII following transient ischemia. Redistribution to the cytosol was revealed on reperfusion. Northern blot showed CaMKII gene expression decreased by ischemia. Furthermore, autoradiography and confocal immunohistochemical findings provided autophosphorylation of CaMKII in the cytosol, ischemia causing decrease, with gradual recovery on reperfusion. These results indicate a transient partial translocation of CaMKII accompanied by kinase activity, with residual myocardial CaMKII undergoing autophosphorylation during ischemia and reperfusion, demonstrating two different characteristic dynamics of CaMKII.     

Identification of putative voltage-dependent Ca2+-permeable channels involved in cryptogein-induced Ca2+ transients and defense responses in tobacco BY-2 cells

Ca(2+) is the pivotal second messenger for induction of defense responses induced by treatment of pathogen-derived elicitor or microbial infection in plants. However, molecular bases for elicitor-induced generation of Ca(2+) signals (Ca(2+) transients) are largely unknown. We here identified cDNAs for putative voltage-dependent Ca(2+)-permeable channels, NtTPC1A and NtTPC1B, that are homologous to TPC1 (two pore channel) from suspension-cultured tobacco BY-2 cells. NtTPC1s complemented the growth of a Saccharomyces cerevisiae mutant defective in CCH1, a putative Ca(2+) channel, in a low Ca(2+) medium, suggesting that both products permeate Ca(2+) through the plasma membrane. Cosuppression of NtTPC1s in apoaequorin-expressing BY-2 cells resulted in inhibition of rise in cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) in response to sucrose and a fungal elicitor cryptogein, while it did not affect hypoosmotic shock-induced [Ca(2+)](cyt) increase. Cosuppression of NtTPC1s also caused suppression of cryptogein-induced programmed cell death and defense-related gene expression. These results suggest that NtTPC1s are involved in Ca(2+) mobilization induced by the cryptogein and sucrose, and have crucial roles in cryptogein-induced signal transduction pathway.