Hiromi Hayashi

Methylmercury induces Ca2+-dependent hyperpolarization of mouse thymocytes: a flow cytometric study using fluorescent dyes

The effect of methylmercury on mouse thymocytes was examined using fluorescent dyes for membrane potential and intracellular Ca2+. Methylmercury at concentrations of 1 microM or higher (up to 30 microM) produced hyperpolarization in a dose-dependent fashion. Charybdotoxin and quinine, but not 4-aminopyridine and tetraethylammonium, greatly suppressed methylmercury-induced hyperpolarization. Removal of external Ca2+ reduced the degree of hyperpolarization. Pretreatment of thymocytes with A23187 under Ca(2+)-free conditions abolished the hyperpolarization induced by methylmercury. Under both normal and Ca(2+)-free conditions methylmercury increased the intracellular concentration of Ca2+. The results suggest that the increase in intracellular Ca2+ is mediated through a Ca2+ release from intracellular stores as well as through influx of external Ca2+. Therefore, it is likely that methylmercury increases the intracellular concentration of Ca2+, resulting in activation of Ca(2+)-dependent K+ conductance of mouse thymocytes.

Toxicity of methylmercury conjugated with L-cysteine on rat thymocytes and human leukemia K562 cells in comparison with that of methylmercury chloride.

In order to reveal the implication of use of methylmercury chloride (MeHgCl) in in vitro study, the effects of 10 µM MeHgCl on rat thymocytes and human leukemia K562 cells were compared with those of methylmercury conjugated with L-cysteine (10 µM MeHg-Cys) using a flow cytometer and fluorescent probes to monitor cellular physiological and pathological parameters. MeHgCl hyperpolarized membranes of thymocytes, followed by depolarization within a few minutes after the application, while MeHg-Cys persistently hyperpolarized them. MeHgCl increased intracellular concentration of Ca(2+), decreased cellular content of glutathione and increased generation of superoxide anion in the cells. The effects of MeHg-Cys were much less than those of MeHgCl. MeHgCl greatly increased both numbers of the cells undergoing apoptosis and dead cells in cell suspension containing thymocytes, while this was not the case for MeHg-Cys. MeHgCl reduced the cell viability of human leukemia K562 cells and completely inhibited the cell growth. The effects of MeHg-Cys on K562 cells were less than those of MeHgCl. It can be concluded that the effects of MeHgCl on rat thymocytes and K562 cells are different from those of MeHg-Cys. The results obtained from the in vitro studies using MeHgCl may be less implicit to elucidate the mechanism of MeHg intoxication in humans and experimental animals because MeHg are present in forms of MeHg-Cys and/or MeHg-S conjugate under the in vivo conditions.

Isolation of a novel lectin from the globiferous pedicellariae of the sea urchin Toxopneustes pileolus.

Sea urchin lectin-I (SUL-I), a 32 kDa lectin was purified from the large globiferous pedicellariae of the sea urchin, Toxopneustes pileolus by using gel permeation chromatography, ion-exchange chromatography and reverse-phase HPLC. SDS-PAGE showed that SUL-I is a monomeric protein with a molecular mass of 32 kDa. Amino acid analysis indicates SUL-I to contain 294 residues. SUL-I was shown to have chemotactic properties for guinea-pig neutrophils at concentrations of 0.625 microgram/ml. These data suggest that a 32 kDa lectin from T. pileolus may be related to defensive role.

Isolation and characterization of a neutrophil chemotactic factor from Tritrichomonas foetus organisms

A neutrophil chemotactic factor (TfNCF) was isolated from the crude extract of Tritrichomonas foetus organisms by a combination of anion‐exchange chromatography on DE52 and gel filtration on Sephacryl S200. TfNCF showed homogenicity by both PAGE and SDS‐PAGE. The molecular weight of TfNCF was estimated to be 22 and 24 kDa, by Sephacryl S200 gel chromatography and by SDS‐PAGE under reducing conditions. Immunization of TfNCF caused almost complete protection against T. foetus infection in mice. Western blot analysis probed with anti‐TfNCF antibody showed that the epitopes on TfNCF were not commonly shared on the other components of T. foetus organisms nor other helminthous parasite‐derived components. Furthermore, pre‐incubation of neutrophils with antigens of other helminthous parasites or N‐formylmethionyl‐leucyl‐phenylalanine did not affect the neutrophil chemotactic activity for TfNCF. These results suggest that TfNCF is a novel NCF consisting of unique epitopes for both antigenicity and neutrophil chemotactic activity. The role of NCF in the initiation of the immune response is discussed.

Seasonal changes in contractile activity of a toxic substance from the pedicellaria of the sea urchin Toxopneustes pileolus

The seasonal change in the activity of an extract from the pedicellariae of the sea urchin Toxopneustes pileolus was investigated in terms of contraction of the longitudinal muscle of the isolated guinea-pig ileum. The protein level of the crude extract from the pedicellariae was high in May, June and July, but was not parallel to the activity of the extract which was low in summer (0.03 X 10(3)-0.33 X 10(3] and became higher in autumn and winter (0.51 X 10(3)-1.1 X 10(3]. The changes in protein level and the specific activity of the extract were also not closely correlated to the reproductive season of the sea urchins. The specific activity of Peak II fraction purified from the extract was high in spring (May), lowest in summer and elevated again in autumn and winter.

d -Allose and d -psicose reinforce the action of metronidazole on trichomonad

The effects of d-allose and d-psicose on Tritrichomonas foetus were examined. They were cultured in F-bouillon medium including glucose, but had never increased when glucose was substituted to those sugars. When cultured in a medium including a dose of ED50 metronidazole and those sugars, trichomonad density was significantly less than that in a medium with metronidazole only. d-Allose remarkably reinforced the action of metronidazole. This means there are some interactions between metronidazole and those sugars. Although the mechanism is not clear, by using those sugars for treatment with metronidazole, the drug dosage could be lowered and the development of drug resistance of trichomonad parasites might be prevented.

Effects of A23187 and CaCl2 on tri-n-butyltin-induced cell death in rat thymocytes

As tri-n-butyltin (TBT), one of the environmental pollutants, is accumulated in wild animals, concern regarding the toxicity of TBT in both wildlife and human is increasing. TBT has been reported to increase intracellular Ca(2+) concentration in several types of cells. In order to examine how Ca(2+) is involved in TBT-induced cell death, the effect of TBT on rat thymocytes has been compared with that of A23187, a calcium ionophore, under various concentrations of external Ca(2+) using a flow cytometer and fluorescent probes. Although both TBT and A23187 were toxic to cells under normal Ca(2+) condition, under external Ca(2+)-free condition the cytotoxic action of TBT was potentiated without changing the threshold concentration while that of A23187 was completely abolished. A23187 attenuated the TBT-induced descent in cell viability under normal Ca(2+) concentration despite intracellular Ca(2+) concentration was increased. As external Ca(2+) concentration increased, the TBT-induced increase in number of dead cells gradually decreased whereas the number of cells in an early stage of apoptosis increased. Results suggest that Ca(2+) has contradictory actions on the process of TBT-induced cell death in rat thymocytes.

Cytokinesis arrest and nuclear fission in low density populations of trichomonad protozoan

Abstract Cell growth of anaerobic protozoan Tritrichomonas foetus was analyzed. This protozoan usually proliferates in extremely high density, but protozoan parasites were dispersed uniformly in F-bouillon medium and cell division stopped temporarily. However, nuclear fission continued and giant polynucleated cells formed. Later, cell division resumed and cells returned to normal form. In conditioned medium, cytokinesis of the dispersed parasites did not stop. Results indicated that T. foetus cells secreted an extra-cellular factor that influenced cytokinesis.

Effects of Dextran Sulfate 500 on Protective Responses to Sublethal Trichomonas foetus Infection in Mice

In order to analyze the host‐parasite interactions in experimental trichomoniasis, the growth of Trichomonas foetus in the peritoneal cavity and changes in the peritoneal exudate cells were followed in mice treated with dextran sulfate 500 (DS 500), a known macrophage‐toxic agent. Light microscopic observation showed that DS 500 treatment induced degeneration of peritoneal macrophages within about 48 hr after the treatment and the damaged macrophages did not phagocytize the parasites, whereas peritoneal neutrophils and lymphocytes were not affected by the drug. In the DS 500‐treated mice, growth of parasites in the peritoneal cavity was accelerated and a high susceptibility of the mice to T. foetus infection was observed. These results indicate that macrophages play the most important role among the peritoneal exudate cells in resistance to T. foetus infection, especially during the early stage of infection.