The migratory gametic nuclear exchange is an important event during the conjugation of ciliates. So far, it is unclear whether this process occurs in Paramecium polycaryum, and there are also some other contradictory descriptions of this species. To clarify this issue, we studied the conjugation process in the Chinese strain of P. polycaryum by means of a modified protargol technique. The reciprocal gametic nuclear exchange and the formation of fertilized nuclei were observed in detail. The results indicated the following: 1) P. polycaryum has cross-fertilization during its conjugation. 2) The transfer of migratory gametic nuclei into the partner cell seems to be an active process. 3) The process of fertilized nuclear formation includes the attachment of a migratory gametic nucleus to a stationary one, membrane fusion of two kinds of gametic nuclei with separate nucleoplasm, and the fusion of the nucleoplasm of two gametic nuclei to form a typical fertilized nucleus.

During conjugation of Paramecium caudatum, four haploid nuclei are formed after meiosis, only one of those that enters the paroral region survives and divides once more to form two gametic nuclei, and the remaining three degenerate. Here, we applied the method of co-staining the living cells with acridine orange and Hoechst 33342, which had been found effective to identify the apoptotic nuclei in Tetrahymena, to clarify whether the meiotic nuclear degeneration in P. caudatum is an apoptotic process. We found that three haploid nuclei outside the paroral region were stained green-yellow, while the survivor in the paroral region was stained blue. When the conjugating pairs separated and the synkaryon divided twice, three degenerating haploid nuclei were still observed and stained yellow. The old fragmented macronuclei, which are known to be still alive in exconjugants, were stained blue, in sharp contrast with the case in Tetrahymena. These results strongly suggest that the haploid nuclei degenerate in an apoptotic way in P. caudatum.

In Blepharisma japonicum, conjugation is induced by interaction between cells of complementary mating-types I and II. Cells of both mating-types produce and secrete mating pheromones (gamones). Cells that have received complementary gamones undergo morphological changes (rounding) and start to unite. Nuclear changes, including meiosis, gametic nuclei exchange, fertilization and development of new macro- and micronuclei occur in the conjugants. Although conjugation is such a striking phenomenon, the molecular mechanism of induction of conjugation remains unknown. In order to identify genes that are involved in formation of conjugating pairs, we isolated genes that were expressed specifically in conjugation-induced type II cells, using suppression subtractive hybridization. To induce conjugation, we treated type II cells for 4 hours with a cell-free fluid from type I cells which contained gamone1. During this period, type II cells formed pairs, although these homotypic pairs never entered meiosis. We then purified polyA⁺RNA and subjected it to cDNA synthesis. This cDNA was then subtracted with cDNA that was prepared from untreated cells. We obtained eight gene fragments. Homology searches revealed that three of these fragments showed significant homology to the cdk family (cdk1 and cdk2), 4-hydroxy-phenylpyruvate dioxygenase (4-HPPD) and cyclin dependent kinase regulatory subunit (cks). Northern hybridization demonstrated that these three genes were specifically transcribed in cells treated with gamone1. We also found that the transcripts had already appeared 2 hours after the onset of gamone1 treatment. Cdk1 and cks are generally involved in cell-cycle regulation, but are here specifically expressed during induction of homotypic type II pairs that undergo neither mitosis nor meiosis.

Carbon nanofibers (CNF), composed of carbon nanotubes, are a recent technological advance with wide applications in nano-engineering fields including biotechnology and biomedicine. However, little is known about the environmental effects of CNF, or their potential danger to human health. To elucidate the safety of CNF, we examined the cytotoxicity of CNF in Paramecium. In this study we considered the cytotoxicity effect of CNF in two categories of cellular properties, cell survival and cell proliferation. We show that CNF are ingested and concentrated as efficiently as nutritive bacteria by paramecia, revealing a means by which CNF could be introduced into the food webs of aquatic ecosystems. Clear cytotoxicity of CNF was detected in survival tests by extracellular application at extremely high concentration (up to 50 mg/ml) in culture medium containing nutritive bacteria. Contrary to this effect, no cytotoxicity was detected in survival tests using the modified Dryl's solution (K-DS) that is used as a buffered saline of culture medium. The cytotoxicity of CNF suggests an interaction between CNF and the components in culture medium or their metabolic products produced by digestion of components in culture medium. Another cytotoxicity effect was detected in proliferative activity test at lower concentrations of CNF (up to 500 µg/ml). The cytotoxicity on proliferative activity was reversible and recovery occurred within 24 hours after removal of CNF. Our results suggest that Paramecium is useful for a bioassay of nanoparticle cytotoxicity. For the elucidation of safety of CNF we have to examine both the optimum concentration of CNF and co-existing biomaterials in a test solution. In conclusion, CNF have a high potential for cytological and biomedical application under precise control of concentration.

Myosin is an eukaryotic motor protein interacting with actin filaments. It is categorized into 24 classes by based on the conserved motor domain. Each myosin possesses divergent functions via characteristic domains in a tail. Most of the intercellular functions of myosins have been studied in metazoan cells. For example, myosins function in cytokinesis, phagocytosis, cell migration and cargo transport. In addition, some myosins are involved in regulating signal transduction. It has recently been uncovered that several novel types of myosins function in Protozoa and exert in unique cellular function. In this mini review I would like to introduce the attraction of protozoan myosins.

The contractile vacuole (CV) is an organelle particularized to osmoregulation in protozoa. However, the understanding of mechanism of water uptake is still poor. The aim of the present article is to review the mechanism of water accumulation by CV in Amoeba proteus.

To visualize the dynamics of the CV in living cell, we used a styryl dye, FM4–64. Just after systole, the CV membrane was flattened. During diastole, a few vesicles were formed from the membrane and they fused each other before reformation of CV. Staining was maintained during continued contraction cycles. We conclude that the CV membrane is maintained during the CV cycle.

Based on the water permeability, the presence of aquaporin in the CV membrane has been predicted and we succeeded in cloning an aquaporin gene from A. proteus (ApAQP). Immunofluorescence microscopy with anti-ApAQP antibody revealed that ApAQP was localized on the CV membrane and the vesicles around CV. This is the first success in explaining high water permeability of the CV membrane by aquaporin. In addition, we found that V-ATPase was highly concentrated on the vesicle membranes around CV. These finding suggest that vesicles are involved in generating the osmotic gradient via the activity of V-ATPase and that water moves into the vesicles along the osmotic gradient.