The dying process of Paramecium tetraurelia cells under a cover glass without supporting pillars was described. The initiation of the process was defined by the cessation of swimming, and the termination by cell rupture. The process required 6 min on average, ranging from 1 to 11 min. The first symptom of death was the formation of a small bleb, a local swelling of the outer cell membrane. The number of blebs increased, each bleb grew, and neighboring blebs fused to form a larger bleb. By supplying water, the blebs disappeared only when their size and numbers were relatively small, indicative of a commitment point to death. Finally the outer membrane and then the inner membrane were broken, and the cytoplasmic contents flew out. Until the last moment of the cell rupture, and sometimes even after that, ciliary beating was observed somewhere locally on the cell membrane. Among the cilia, those at the cytopharynx were the first to stop beating so that food vacuole (FV) formation stopped somewhat earlier than the cellular disorganization. Cessation of the contractility of contractile vacuoles (CVs) occurred a little bit later on average than the cessation of FV formation. Anterior and posterior CVs were not related to each other in the timing of loss of contractility and in the preceding pulsating cycles. Finally, we calculated the physical pressure to burst the cell at about 950 atm and estimated the actual pressure to be at 470～2,000 atm.
In this study, we revealed that the lectin wheat germ agglutinin (WGA) and its specific binding sugars, N-acetylneuraminic acid (NeuAc) and N-acetylglucosamine (GlcNAc), inhibited mating pair formation in Paramecium caudatum. The concentrations that caused 50% inhibition (IC50) were 17 nM, 3 mM and 30 mM, respectively. Using FITC-WGA, it was shown fluorescence-cytochemically that WGA bound to the adhesion site between conjugated cells; this binding was inhibited by NeuAc but not GlcNAc. Moreover, the binding of FITC-WGA was maintained appreciably when its localization was changed in correspon-dence with progression of conjugation until exchange of gametic nuclei was completed at which point the binding between both cells disintegrated. These results strongly suggested that NeuAc plays a primary role in mating pair formation in P. caudatum as a specific WGA binding sugar.
Resent insights into the origin and early evolution of mitochondria come from two approaches: the investigation of mtDNAs from minimally derived (primitive) mitochondriate eukaryotes, in particular jakobid flagellates, and of genomes from intracellular α-proteobacterial symbionts. Of particular interest in this context is Holospora obtusa, an intracellular bacterial endosymbiont that resides and replicates in the somatic nucleus of its eukaryotic host, the ciliate Paramecium caudatum. Currently we have sequenced close to 50% of the ～1.7 Mbp H. obtusa genome, revealing the absence of genes for oxidative phosphorylation, the TCA cycle, and many other metabolic pathways, but the presence of several pathogenesis-related genes and a high number of bacterial IS elements. Phylogenetic analyses with multiple protein sequences place H. obtusa basally to the Rickettsia-Ehrlichia-Wolbachia assemblage of bacterial pathogens. This leads us to postulate that H. obtusa is the closest bacterial relative of mitochondria known to date.