Yukishige Kawasaki

Visualization of exocytotic secretory processes of mast cells by fluorescence techniques

Secretory processes via exocytosis in rat peritoneal mast cells were visualized by two complementary fluorescence techniques; one staining pre-exocytotic granules with a basic probe and the other staining post-exocytotic granules with acidic probes. Granules within mast cells were selectively stained with acridine orange and emitted orange yellow fluorescence. Upon stimulation with compound 48/80, release of acridine orange from granules was observed both in population and single cell measurements. This release was seen in some localized area of mast cells. Opening of pores between plasma membranes and granule membranes was monitored using acidic fluorescence probes such as 6-carboxyfluorescein or lucifer yellow CH. Not only granules located at peripheral region, but also granules near the core region participated in exocytosis. The existence of junctions between these granules was suggested. TMA-DPH, a lipophilic membrane probe, which was localized at plasma membrane before stimulation, diffused into granule membranes after stimulation. This shows that after stimulation, some constituents of plasma and granule membranes were mixed. Even after extensive degranulation, mast cells extruded acidic probes, indicating the plasma membranes still play a role of barrier. Activation of lateral motion of granules preceding to exocytosis was not observed. It was concluded that the visualization of secretory processes by fluorescence and image processing techniques will be useful for the study of molecular mechanisms underlying exocytosis.

A change in membrane microviscosity of mouse neuroblastoma cells in association with morphological differentiation.

Changes in membrane microviscosity as well as in membrane constituents of mouse neuroblastoma clone N-18 were studied in association with neurite formation. The membrane microviscosity studied by fluorescence technique increased with the formation of neurites. The concomitant increase increase in the ratio of cholesterol to phospholipids was also observed.

Studies in the search for life on Mars.

The ability of living organisms to survive extraterrestrial conditions has implications for the origins of life in the solar system. We have therefore studied the survival of viruses, bacteria, yeast, and fungi under simulated Martian conditions. The environment on Mars was simulated by low temperature, proton irradiation, ultraviolet irradiation, and simulated Martian atmosphere (CO2 95.46%, N2 2.7%, water vapor 0.03%) in a special cryostat. After exposure to these conditions, tobacco mosaic virus and spores of Bacillus, Aspergillus, Clostridium, and some species of coccus showed significant survival.

Direct detection of Martian microorganisms based on fluorescence microscopy

Abstract The direct detection of microorganisms and their traces using optical microscopes is one of the most promising techniques to obtain the decisive evidences for extraterrestrial life. The most significant points of this technique are high sensitivity and spatial information with a resolution of 0.2mm. Besides, information on local environments and microscopic ecology can also be obtained. Many difficulties, however, must be solved to get reliable results. We have started to develop a noble technique based on the fluorescence microscopy with special interest to the detection of microorganisms in extreme environments including Mars. The principle is to detect molecules/subcellular organs which are responsible for the three basic characteristics of life; genetic information, metabolism, and discrimination of self from non-self. We have screened fluorescence probes and found several are applicable. We could detect almost all the microorganisms already identified. Discrimination of viable from dead cells was possible. The terrestrial microfossils, some of the artificial primitive microorganism-like-objects, dried bacteria and polycyclic aromatic hydrocarbons mixed with simulated Martian sand could be detected. We are now designing a compact detection hardware.

The maturation-dependent change in fibronectin receptor density of mouse dorsal root ganglion neurons

The maturation-dependent change in fibronectin receptor density of mouse dorsal root ganglion neurons were investigated by an immuno-cytofluorometric method. The receptor density showed a drastic decrease around birth and a smaller change after birth.

A mathematical model for microcosms: formation of the colonies and coupled oscillation in population densities of bacteria

A microcosm constructed, in the present study, consisted of bacteria (five or more species), green algae and rotifers and maintained a steady state for more than 100 days. This microcosm formed numerous colonies at the bottom of the flask and demonstrated coupled oscillation in the population densities of bacteria. The population dynamics and the spatial patterns of organisms were simulated using a cellular automata method. From the calculation results, the following facts were inferred: (1) heterogeneous metabolite concentrations due to the diffusion phenomenon as well as the frequencies of interactions among rotifers, bacteria and green algae and the strengths of their interactions are the contributing factors to the colony formation; (2) the system becomes stable when colonies are formed; and (3) coupled oscillation occurres in the population densities of bacteria when certain combinations are selected for the specific growth rate coefficients of the coexisting bacteria, and the distribution of the ratios of specific growth rate coefficients of the bacteria which can coexist in a stable manner is discrete rather than continuous.

Fundamental studies concerning planetary quarantine in space

If there is a possibility that the organisms carried from Earth to space can live for a significant period on planets, the contamination of planets should be prevented for the purpose of future life-detection experiments. In connection with quarantine for interplanetary missions, we have examined the survivabilities of terrestrial microorganisms under simulated space conditions. In this study, examined the survivabilities of terrestrial organisms under simulated Mars conditions. The Mars conditions were simulated by ultraviolet (UV) and proton irradiation under low temperature, high vacuum, and simulated gaseous conditions. After exposure to the simulated Mars condition, the survivabilities of the organisms were examined. The spores of Bacillus subtilis and Aspergillus niger, some anaerobic bacterias and algaes, showed considerably high survivabilities even after UV and proton irradiation corresponding to 200 years on Mars. This subject is not restricted to academic curiosity but concerns problems involving the contamination of Mars with terrestrial organisms carried by space-probes.

Lateral diffusion of membrane lipids changes with aging in C57BL mouse dorsal root ganglion neurons from a fetal stage to an aged stage

The membranes of dorsal root ganglion (DRG) neurons dissected from different age groups of C57BL mice, from 18-day fetal to 30-month-old mice, were labeled with the fluorescent analog of fatty acids, F18, after a day in culture. The fluorescent probe specifically labeled the cell surface. The lateral diffusion of F18 (5-(octadecylthiocarbamoylamino) fluorescein) was measured with fluorescence photobleaching recovery (FPR) method. During development, the lateral diffusion coefficients of F18 rapidly decreased from (0.34 +/- 0.07) X 10(-8) cm2/s (18-day-old fetus) to (0.22 +/- 0.07) X 10(-8) cm2/s (3-day-old newborn). Then the values slowly decreased and reached (0.13 +/- 0.05) X 10(-8) cm2/s in a 6-month-old stage. In stages older than 6-months the lateral diffusion coefficients scarcely changed with aging. As this decrease in the membrane fluidity with increasing age is parallelled to that in capacities of extending neurites, it is thought that the membrane fluidity might change with aging in concert with changes in important cell functions.

Cell membrane expansion and blockade of action potentials produced by 2-decenoic acid in cultured dorsal root ganglion neurons

2-Decenoic acid, a fatty acid having 10 carbon atoms, blocks the action potentials of cultured dorsal root ganglion (DRG) neurons and this effect of 2-decenoic acid is reversible. From the analysis of the video pictures from Nomarski optics, relative values of the diameter and the thickness of the neurons increased to 1.06 and 1.14, respectively, when 2.1 mM 2-decenoic acid was applied to the neurons. The relative value of cell surface area, which was calculated from the equation for a spheroid, increased to about 1.20. On the other hand, relative fluorescence intensity of the fluorescent probe F18 (5-(octadecylthiocarbamoylamino)fluorescein) labeled neurons decreased to 0.81, when 2.1 mM 2-decenoic acid was applied to the neurons. This indicates that the relative cell surface area increased to 1.23, a value similar to that calculated from the results of the measurement of cell size. The time course of blocking action potentials after treatment of the fatty acid was similar to that of the cell membrane expansion. These results show that the fatty acid perturbs the cell membrane and expands the cell surface area and this expansion might reduce the opening ability of the Na+-channels in the membrane.

Behavior of Japanese tree frogs under microgravity on MIR and in parabolic flight.

Japanese tree frogs (Hyla japonica) were flown to the space station MIR and spent eight days in orbit during December, 1990. Under microgravity, their postures and behaviors were observed and recorded. On the MIR, floating frogs stretched four legs out, bent their bodies backward and expanded their abdomens. Frogs on a surface often bent their neck backward and walked backwards. This behavior was observed on parabolic flights and resembles the retching behavior of sick frogs on land--a possible indicator of motion sickness. Observations on MIR were carried out twice to investigate the frogs adaptation to space. The frequency of failure in landing after a jump decreased in the second observation period. After the frogs returned to earth, readaptation processes were observed. The frogs behaved normally as early as 2.5 hours after landing.