Yasuo Nakaoka

EXTRACTION OF RETINAL FROM Paramecium bursaria

Abstract— The ciliated protozoan Paramecium bursaria is photosensitive. Retinal, which is the chromophore of the visual pigments of many multicellular animals, was extracted from P. bursaria and identified by HPLC. The procedure of identification was based on the change in polarity achieved by reducing retinal with sodium borohydrate to form retinol. This method is useful for the detection of small amounts of retinal in cells containing large amounts of other substances with similar polarity. The presence of retinal in P. bursaria suggests that this ciliate may contain a photoreceptor pigment with retinal as the chromophore.

Behavior of micro-organisms as particles with internal state variables

Abstract A theory is presented to describe the behavior of micro-organisms, bacteria and protozoa. Individual cells are regarded as particles having internal state variables. The change of each variable with time depends on the environmental condition. The velocity and the frequency of direction change of swimming cells are determined by the values of these variables. With this framework, the theory gives a method to connect the behaviour in a spatial gradient of the environment and the behaviour upon a change of the environment with time. Observed behaviors of bacteria and protozoa are understandable on the basis of simple rate equations for internal state variables and the product expressions for the velocity and the frequency of direction change as functions of these variables. Experimental data on the thermotaxis of paramecium are shown for comparison with the theoretical results.

Calcium regulates independently ciliary beat and cell contraction in Paramecium cells

Intracellular Ca(2+) concentration is a well-known signal regulator for various physiological activities. In many cases, Ca(2+) simultaneously regulates individual functions in single cells. How can Ca(2+) regulate these functions independently? In Paramecium cells, the contractile cytoskeletal network and cilia are located close to each other near the cell surface. Cell body contraction, ciliary reversal, and rises in ciliary beat frequency are regulated by intracellular Ca(2+) concentration. However, they are not always triggered simultaneously. We injected caged calcium into Paramecium caudatum cells and continuously applied weak ultraviolet light to the cells to slowly increase intracellular Ca(2+) concentration. The cell bodies began to contract just after the start of ultraviolet light application, and the degree of contraction increased gradually thereafter. On the other hand, cilia began to reverse 1.4s after the start of ultraviolet application and reversed completely within 100ms. Ciliary beat frequency in the reverse direction was significantly higher than in the normal direction. These results indicate that cell body contraction is regulated by Ca(2+) in a dose-dependent manner in living P. caudatum. On the other hand, ciliary reversal and rise in ciliary beat frequency are triggered by Ca(2+) in an all-or-none manner.

Hyperpolarization-activated inward current associated with the frequency increase in ciliary beating of Paramecium

SummaryIn order to study the relationship between the inward Ca current activated by hyperpolarization and the frequency increase in ciliary beating, Paramecium cells were voltage clamped under conditions where K current was suppressed by use of CsCl electrodes and by extracellular tetraethyl ammonium. A 2-s pulse of hyperpolarization from the resting potential activated an inward current consisting of two components, an initial transient current peaking at 0.1–0.2 s (which had been identified as a Ca current) and a subsequent sustained current. The initial component was not associated with the frequency increase because the frequency increase was normally induced even when the peak current was almost completely inhibited by external addition of Ba2+. The second sustained current was closely correlated with the frequency increase. The frequency rose steeply with the sustained current and saturated at −0.6 nA. External addition of La3+ or replacement of Ca2+ by Mg2+ suppressed this current, and at the same time the frequency increase was inhibited. As the amplitude of the sustained current was not changed by deciliation, this current must pass through the somatic membrane. These results suggest that the frequency increase upon hyperpolarization is triggered by the voltage-activated inward current passing through the somatic membrane of the interciliary compartment.

Localization of photosensitivity inParamecium bursaria

Summary1.Paramecium bursaria was stimulated by a light spot of 10–15 μm diameter, and the photosensitive site was searched by recording responses in swimming behavior and in membrane potential.2.Local stimulation to the anterior half of the cell caused an avoiding response.3.Stimulation to the cells deciliated by ethanol treatment elicited a depolarization of the membrane potential.4.Local stimulation to the anteroventral portion elicited a depolarization, but stimulation to the dorsal side induced no change in the membrane potential.5.The action spectrum of depolarization elicited by local stimulation to the anteroventral surface showed two main peaks at 420 nm and 560 nm, corresponding to those of light stimulation of the whole cell.6.It is concluded that a photosensitive site exists on the anteroventral surface ofParamecium, in particular within the oral groove of the cell. This local photosensitivity is discussed with respect to the mating reaction.

COLD-SENSITIVE CA2+ INFLUX IN PARAMECIUM

Abstract. The concentration of intracellular calcium, [Ca2+]i, in Paramecium was imaged during cold-sensitive response by monitoring fluorescence of two calcium-sensitive dyes, Fluo-3 and Fura-Red. Cooling of a deciliated Paramecium caused a transient increase in [Ca2+]i at the anterior region of the cell. Increase in [Ca2+]i was not observed at any region in Ca2+-free solution. Under the electrophysiological recording, a transient depolarization of the cell was observed in response to cooling. On the voltage-clamped cell, cooling induced a transient inward current under conditions where K+ currents were suppressed. These membrane depolarizations and inward currents in response to cooling were lost upon removing extracellular Ca2+. The cold-induced inward current was lost upon replacing extracellular Ca2+ with equimolar concentration of Co2+, Mg2+ or Mn2+, but it was not affected significantly by replacing with equimolar concentration of Ba2+ or Sr2+. These results indicate that Paramecium cells have Ca2+ channels that are permeable to Ca2+, Ba2+ and Sr2+ in the anterior soma membrane and the channels are opened by cooling.

The Photodynamic Action of Methylene Blue on the Ion Channels of Paramecium Causes Cell Damage

Abstract— The photodynamic effects of methylene blue (MB) on wild‐type and mutant strains of Paramecium Were studied. From measurements of survival and cell motility under the continuous application of light in the presence of MB, the mutant strains remained alive for about three times longer than the wild‐type strain. Although the resting potential of the mutant cells was similar to that of wild‐type cells, the continuous photodynamic action shifted the membrane potentials of the mutant and wild‐type cells to a depolarized level and a hyperpolarized level, respectively, from that before light application. Under voltage clamping, the mutant cells reduced not only the outward current elicited by the photodynamic action but also the outward tail current elicited by the preceding pulse of hyperpolarization. We conclude that the mutant strain is defective in the activation of Ca2+‐dependent K+channels. This defect might cause a reduction in the Ca2+influx because of the suppression of the membrane hyperpolarization, which results in the elongation of survival time under the photodynamic action.

A calcium-activated, large conductance and non-selective cation channel in Paramecium cell

A non-selective cation channel was found in mutant Paramecium cells (K115). This cell had been selected as a resistant mutant in a high-K+ solution. In patch clamp studies of these cells in the inside-out configuration, this channel was activated by bath applications of elevated Ca2+ concentrations. The channels became very active when the Ca2+ concentration was above 3.2 microM. The channel was also activated by depolarization. The voltage dependency was steep upon depolarization, whereas upon hyperpolarization the channel activity barely changed. This channel had poor selectivity for monovalent alkali cations. Using the Goldman-Hodgkin-Katz equation for the reversal potential, the permeability ratios with respect to K+ for Na+, Rb+, Cs+ and Li+ were nearly 1. Although the permeability ratios were similar for each cation, the single channel conductances differed. The single channel conductances were 467 pS with K+ as the charge carrier, 406 pS with Na+, 397 pS with Rb+, 253 pS with Cs+ and 198 pS with Li+ upon depolarization in 100 mM cation solutions. A similar calcium-activated large conductance channel was observed in the wild-type (G3) Paramecium cells but was very rare.

Cloning of a novel ubiquitin-conjugating enzyme (E2) gene from the ciliate Paramecium tetraurelia☆

We isolated a 1.7 kb gene (UbcPl) for a ubiquitinconjugating enzyme from a P. tetraurelia cDNA library and sequenced it. Its deduced polypeptide sequence consists of 425 amino acid residues (48 kDa). The UbcP1 protein contains novel N‐ and C‐terminal extensions in addition to a UBC domain, and within the UBC domain it shares low identity with sequences of other known E2s. A constructed phylogenetic tree suggests that the UbcP1 protein may represent a member of a distinct subfamily of E2s. Southern blot analysis showed that the N‐terminal extension of the UbcP1 is conserved in P. multimicronucleatum.

Effect of a 60 Hz magnetic field on the behavior of Paramecium.

Paramecium multimicronucleatum was used as a model cell to study the effects of 60 Hz magnetic fields on swimming behavior. When exposed to a vertical field of 0.6 T, the cells accumulated at the upper end of the cuvette. An analysis of the swimming behavior revealed that the exposure to the field increased the number of cells swimming upwards maximally at 1 min after onset of the exposure. This effect of the magnetic field was transient, disappearing within a few minutes during the exposure. It is suggested that the magnetic field may amplify to a large extent the negative gravitaxis of Paramecium. Effects of an induced electric field on the swimming behavior are also discussed.