Young's interference of electrons in field emission patterns.

We have observed interference fringes of electrons in field emission patterns from multiwalled carbon nanotubes at 60 K. The observed fringe pattern is reproduced by calculations based on the formula of Young's interference of two beams. Three-beam interference has also been detected over short time periods. We discuss the reason why Young's interference appears in the electron emission pattern in accelerating fields.

A new learning algorithm for the hierarchical structure learning automata operating in the nonstationary S-model random environment.

An extended algorithm of the relative reward strength algorithm is proposed. It is shown that the proposed algorithm ensures the convergence with probability I to the optimal path under the certain type of nonstationary environment. Several computer simulation results confirm the effectiveness of the proposed algorithm.

Mechanisms underlying extracellular ATP-evoked interleukin-6 release in mouse microglial cell line, MG-5.

Microglia play various important roles in the CNS via the synthesis of cytokines. The ATP-evoked production of interleukin-6 (IL-6) and its intracellular signals were examined using a mouse microglial cell line, MG-5. ATP, but not its metabolites, produced IL-6 in a concentration-dependent manner. Although ATP activated two mitogen-activated protein kinases, i.e. p38 and extracellular signal-regulated protein kinase, only p38 was involved in the IL-6 induction. However, the activation of p38 was not sufficient for the IL-6 induction because 2'- and 3'-O-(4-benzoylbenzoyl) ATP, an agonist to P2X7 receptors, failed to produce IL-6 despite the fact that it activated p38. Unlike in other cytokines in microglial cells, P2Y rather than P2X7 receptors seem to have a major role in the IL-6 production by the cells. The ATP-evoked IL-6 production was attenuated by Gö6976, an inhibitor of Ca(2+)-dependent protein kinase C (PKC). The P2Y receptor responsible for these responses was insensitive to pertussis toxin (PTX) and was linked to phospholipase C. Taken together, ATP acting on PTX-insensitive P2Y receptors activates p38 and Ca(2+)-dependent PKC, thereby resulting in the mRNA expression and release of IL-6 in MG-5. This is a novel pathway for the induction of cytokines in microglia.

Theoretical and experimental dissection of gravity-dependent mechanical orientation in gravitactic microorganisms.

Mechanisms of gravitactic behaviors of aquatic microorganisms were investigated in terms of their mechanical basis of gravity-dependent orientation. Two mechanical mechanisms have been considered as possible sources of the orientation torque generated on the inert body. One results from the differential density within an organism (the gravity-buoyancy model) and the other from the geometrical asymmetry of an organism (the drag-gravity model). We first introduced a simple theory that distinguishes between these models by measuring sedimentation of immobilized organisms in a medium of higher density than that of the organisms. Ni2+-immobilized cells of Paramecium caudatum oriented downwards while floating upwards in the Percoll-containing hyper-density medium but oriented upwards while sinking in the hypo-density control medium. This means that the orientation of Paramecium is mechanically biased by the torque generated mainly due to the anterior location of the reaction center of hydrodynamic stress relative to those of buoyancy and gravity; thus the torque results from the geometrical fore-aft asymmetry and is described by the drag-gravity model. The same mechanical property was demonstrated in gastrula larvae of the sea urchin by observing the orientation during sedimentation of the KCN-immobilized larvae in media of different density: like the paramecia, the gastrulae oriented upwards in hypo-density medium and downwards in hyper-density medium. Immobilized pluteus larvae, however, oriented upwards regardless of the density of the medium. This indicates that the orientation of the pluteus is biased by the torque generated mainly due to the posterior location of the reaction center of gravity relative to those of buoyancy and hydrodynamic stress; thus the torque results from the fore-aft asymmetry of the density distribution and is described by the gravity-buoyancy model. These observations indicate that, during development, sea urchin larvae change the mechanical mechanism for the gravitactic orientation. Evidence presented in the present paper demonstrates a definite relationship between the morphology and the gravitactic behavior of microorganisms.

Effect of gravity field on the nonequilibrium/nonlinear chemical oscillation reactions.

Biological systems have evolved for a long time under the normal gravity. The Belousov-Zhabotinsky (BZ) reaction is a nonlinear chemical system far from the equilibrium that may be considered as a simplified chemical model of the biological systems so as to study the effect of gravity. The reaction solution is comprised of bromate in sulfuric acid as an oxidizing agent, 1,4-cyclohexanedione as an organic substrate, and ferroin as a metal catalyst. Chemical waves in the BZ reaction-diffusion system are visualized as blue and red patterns of ferriin and ferroin, respectively. After an improvement to the tubular reaction vessels in the experimental setup, the traveling velocity of chemical waves in aqueous solutions was measured in time series under normal gravity, microgravity, hyper-gravity, and normal gravity using the free-fall facility of JAMIC (Japan Microgravity Center), Hokkaido, Japan. Chemical patterns were collected as image data via CCD camera and analyzed by the software of NIH image after digitization. The estimated traveling velocity increased with increasing gravity as expected. It was clear experimentally that the traveling velocity of target patterns in reaction diffusion system was influenced by the effect of convection and correlated closely with the gravity field.

Japanese red-bellied newts in Space--AstroNewt experiment on Space Shuttle IML-2 and Space Flyer Unit.

Biological effects of gravity was examined in embryonic development of Japanese red bellied newt. Two space newt missions were conducted in 1994 and 1995. The Second International Microgravity Laboratory was flown in 1994 as one of the SpaceLab missions. Space Flyer Unit, a Japanese space platform, was delivered to the earth orbit by the third launch of the H-II rocket and retrieved by Space Shuttle in 1996. Female newts were induced to lay eggs in orbit at these two space missions. Eggs were successfully obtained on both missions, and exposed to space environment from its early developmental stages. Morphology of the embryos was found not deviated from those developed on ground, as long as in the images taken in orbit or the examined specimen retrieved to ground. On the other hand, pathological changes were discovered in several organs of the adult newts that returned alive from their space flight.

Nonequilibrium / nonlinear chemical oscillation in the virtual absence of gravity.

The Belousov-Zhabotinsky (BZ) reactions were used as typical examples of a nonlinear system far from equilibrium in connection with biological evolution. The virtual absence of gravity in the present work was given from the free-fall facility of Japan Microgravity Center (JAMIC) in Hokkaido. The reaction solution of BZ reaction was composed of bromate in sulfuric acid, 1,4-cyclohexanedione and ferroin to visualize the time development of patterns of chemical oscillations in the reaction-diffusion system. It is a bubble-free constitution in the aging of the reaction. Therefore, the setup constructed to collect image data via CCD cameras was simplified. The operation sequences of necessary devices were comprised of simple solid state relays which were started by a command from the operation room of JAMIC. The propagation profile of chemical patterns under microgravity of 10(-5) g was collected as image data for 9.8 s, and processed by a software of STM-STS2. In the aqueous solutions, propagation velocity of chemical patterns under microgravity was decreased to 80.9 % of that under normal gravity, owing to suppression of convection. On the other hand, in gel matrix, gravity did not influence the propagation velocity.

Evaluation of gravity-dependent membrane potential shift in Paramecium.

It is still debated whether or not gravity can stimulate unicellular organisms. This question may be settled by revealing changes in the membrane potential in a manner depending on the gravitational forces imposed on the cell. We estimated the gravity-dependent membrane potential shift to be about 1 mV G-1 for Paramecium showing gravikinesis at 1-5 G, on the basis of measurements of gravity-induced changes in active propulsion and those of propulsive velocity in solutions, in which the membrane potential has been measured electrophysiologically. The shift in membrane potential to this extent may occur from mechanoreceptive changes in K+ or Ca2+ conductance by about 1% and might be at the limit of electrophysiological measurement using membrane potential-sensitive dyes. Our measurements of propulsive velocity vs membrane potential also suggested that the reported propulsive force of Paramecium measured in a solution of graded densities with the aid of a video centrifuge microscope at 350 G was 11 times as large as that for -29 mV, i.e., the resting membrane potential at [K+]o = 1 mM and [Ca2+]o = 1 mM, and, by extrapolation, that Paramecium was hyperpolarized to -60 mV by gravity stimulation of 100-G equivalent, the value corrected by considering the reduction of density difference between the interior and exterior of the cell in the graded density solution. The estimated shift of the membrane potential from -29 mV to -60 mV by 100-G equivalent stimulation, i.e., 0.3 mV G -1, could reach the magnitude entirely feasible to be measured more directly.

Longevity in space; experiment on the life span of Paramecium cell clone in space.

Life span is the most interesting and also the most important biologically relevant time to be investigated on the space station. As a model experiment, we proposed an investigation to assess the life span of clone generation of the ciliate Paramecium. In space, clone generation will be artificially started by conjugation or autogamy, and the life span of the cell populations in different gravitational fields (microgravity and onboard 1 x g control) will be precisely assessed in terms of fission age as compared with the clock time. In order to perform the space experiment including long-lasting culture and continuous measurement of cell division, we tested the methods of cell culture and of cell-density measurement, which will be available in closed environments under microgravity. The basic design of experimental hardware and a preliminary result of the cultivation procedure are described.

Super-helix model: a physiological model for gravitaxis of Paramecium.

A new model explaining the gravitactic behavior of Paramecium is derived on the basis of its mechanism of gravity sensing. Paramecium is know to have depolarizing mechanoreceptor ion channels in the anterior and hyperpolarizing channels in the posterior of the cell. This arrangement may lead to bidirectional changes of the membrane potential due to the selective deformation of the anterior and posterior cell membrane responding to the orientation of the cell with respect to the gravity vector; i.e., negative- and positive-going shifts of the potential due to the upward and downward orientation, respectively. The orientation dependent changes in membrane potential, in combination with the close coupling between the membrane potential and ciliary locomotor activity, may allow the changes in swimming direction along the otherwise simple helical swimming path in the following manner: an upward shift of the axis of helical swimming occurs by decreasing the pitch angle due to channel-dependent hyperpolarization in upward-orienting cells, and an upward shift of the swimming helix occurs by increasing the cell's pitch angle due to depolarization in downward-orienting cells. Computer simulation of the model demonstrated that the cell can swim upward along the "super-helical" trajectory consisting of a small helix winding helically along an axis parallel to the gravity vector.

Modification of ciliary beating in sea urchin larvae induced by neurotransmitters: beat-plane rotation and control of frequency fluctuation

The modification of ciliary beating by neurotransmitters in sea urchin larvae at the four-armed pluteus stage was analyzed in terms of the direction of beating and fluctuation in the beat period. Application of dopamine to Pseudocentrotus depressus causes the cilia to turn their beat plane but retain its characteristic planar feature up to the complete 'reversal' of the beat direction. This new type of response was termed the 'beat-plane turning response'. It was also found that neurotransmitters, especially dopamine and serotonin, can modify the length of the beating cycle in P. depressus and Hemicentrotus pulcherrimus. Dopamine decreased and serotonin increased the beat frequency averaged over the ciliated epithelium with the standard deviation from the mean increasing in the presence of dopamine and decreasing with serotonin. The beat-period fluctuation and its modification suggested by this observation was confirmed from measurements of the beating of individual cilia in the presence or absence of these neurotransmitters. Further analysis of the correlation between angular velocity and beat period indicates that variation in the beat period is not controlled by the same processes as those that modulate angular velocity. These findings in sea urchin larvae suggest that both the stability and the direction of ciliary beating is under nervous control.

Frog experiment onboard space station Mir.

Japanese tree frogs (Hyla japonica) showed unique postures and behavior during an 8-day flight to the Russian space station Mir. When floating in the air, the animals arched their back and extended their four limbs. This posture resembles that observed during jumping or parachuting of the animals on the ground. Frog sitting on a surface bent their neck backward sharply, did not fold their hind limbs completely, and pressed their abdomen against the substrate. They walked backwards in this posture. The typical posture resembles that adopted during the emetic behavior process on the ground, although the posture in space lasts much longer. The possible mechanism of induction of this unique posture in orbit is discussed. Frogs in this posture might be in an emetic state, possibly due to motion sickness. Response behavior to some stimuli was observed in orbit. Body color change in response to the background color appeared to be delayed or slowed down. Response behavior to other stimuli showed little change as long as the animal maintained contact with a substrate. Once it left the surface, the floating frog could not control its movements so as to provide coordinated motility for locomotion and orientation. Adaptation to microgravity was observed in the landing behavior after jumping. Readaptation of the frogs to the Earth environment took place within a few hours after return. Postflight histological and biochemical analysis of organs and tissues showed some changes after the 8-day spaceflight. Weakening and density loss in vertebrae was noted. The beta-adrenoreceptor activity of the gastrocnemius was natriuretic decreased. Skin collagen and liver protein synthesis were lowered. The distribution of the atrial factor-like peptides in the brain was changed.

The Frog in Space (FRIS) experiment onboard Space Station Mir: final report and follow-on studies.

The "Frog in Space" (FRIS) experiment marked a major step for Japanese space life science, on the occasion of the first space flight of a Japanese cosmonaut. At the core of FRIS were six Japanese tree frogs, Hyla japonica, flown on Space Station Mir for 8 days in 1990. The behavior of these frogs was observed and recorded under microgravity. The frogs took up a "parachuting" posture when drifting in a free volume on Mir. When perched on surfaces, they typically sat with their heads bent backward. Such a peculiar posture, after long exposure to microgravity, is discussed in light of motion sickness in amphibians. Histological examinations and other studies were made on the specimens upon recovery. Some organs, such as the liver and the vertebra, showed changes as a result of space flight; others were unaffected. Studies that followed FRIS have been conducted to prepare for a second FRIS on the International Space Station. Interspecific diversity in the behavioral reactions of anurans to changes in acceleration is the major focus of these investigations. The ultimate goal of this research is to better understand how organisms have adapted to gravity through their evolution on earth.

AstroNewt: early development of newt in space.

AstroNewt experiment explores the effects of earth gravity on the early development of Japanese red-bellied newt, Cynops pyrrhogaster. Since female newts keep spermatophore in cloaca, fertilized eggs could be obtained without mating. Fertilization of newt's egg occurs just prior to spawning, so that gonadotrophic cues applied to females in orbit leads to laying eggs fertilized just in space. A property of newt being kept in hibernation at low temperature may be of great help for the space experiment carried out with much limited resources. A general outline of the AstroNewt project is shown here in addition to some technical advances for the development of the project. Experimental schemes of two space experiments (IML-2 in summer 1994 and unmanned SFU at the beginning of 1995) are also shown.

Does Paramecium sense gravity?

In order to get an insight into the cellular mechanisms for the integration of the effects of gravity, we investigated the gravitactic behaviour in Paramecium. There are two main categories for the model of the mechanism of gravitaxis; one is derived on the basis of the mechanistic properties of the cell (physical model) and the other of the physiological properties including cellular gravireception (physiological model). In this review article, we criticized the physical models and introduced a new physiological model. Physical models postulated so far can be divided into two; one explaining the negative gravitactic orientation of the cell in terms of the static torque generated by the structural properties of the cell (gravity-buoyancy model by Verworn, 1889 and drag-gravity model by Roberts, 1970), and the other explaining it in terms of the dynamic torque generated by the helical swimming of the cell (propulsion-gravity model by Winet and Jahn, 1974 and lifting-force model by Nowakowska and Grebecki, 1977). Among those we excluded the possibility of dynamic-torque models because of their incorrect theoretical assumptions. According to the passive orientation of Ni(2+)-immobilized cells, the physical effect of the static torque should be inevitable for the gravitactic orientation. Downward orientation of the immobilized cells in the course of floating up in the hyper-density medium demonstrated the gravitactic orientation is not resulted by the nonuniform distribution of cellular mass (gravity-buoyancy model) but by the fore-aft asymmetry of the cell (drag-gravity model). A new model explaining the gravitactic behaviour is derived on the basis of the cellular gravity sensation through mechanoreceptor channels of the cell membrane. Paramecium is known to have depolarizing receptor channels in the anterior and hyperpolarizing receptors in the posterior of the cell. The uneven distribution of the receptor may lead to the bidirectional changes of the membrane potential by the selective deformation of the anterior and posterior cell membrane responding to the orientation of the cell in the gravity field; i.e. negative- and positive-going shift of the potential due to the upward and downward orientation, respectively. The orientation dependent changes in membrane potential with respect to gravity, in combination with the close coupling of the membrane potential and the ciliary locomotor activity, may allow the changes in swimming direction along with those in the helical nature of the swimming path; upward shift of axis of helix by decreasing the pitch angle due to hyperpolarization in the upward-orienting cell, and also the upward shift by increasing the pitch angle due to depolarization in the downward-orienting cell. Computer simulation of the model demonstrated that the cell can swim upward along the "super-helical" trajectory consisting of a small helix winding helically an axis parallel to the gravity vector, after which the model was named as "Super-helix model". Three-dimensional recording of the trajectories of the swimming cells demonstrated that about a quarter of the cell population drew super-helical trajectory under the unbounded, thermal convection-free conditions. In addition, quantitative analysis of the orientation rate of the swimming cell indicated that gravity-dependent orientation of the swimming trajectory could not be explained solely by the physical static torque but complementarily by the physiological mechanism as proposed in the super-helix model.