Temperature-Sensitive Behavior of Paramecium caudatum

SYNOPSIS. The effect of temperature on the behavior of swimming cells of Paramecium caudatum has been investigated by photographic analyses of their tracks in uniform temperature, in temperature gradient, or in temperature changing with time. When the cells were placed in the temperature gradient, the frequency of discontinuous directional changes of cells swimming toward the optimal temperature, the temperature of the culture, was much lower than that of the cells swimming in the opposite direction. This difference in the frequency of directional changes explained the observed accumulation of the cells at - the optimal temperature. When the temperature was suddenly changed toward the optimum, a transient decrease of the frequency of directional changes was observed and when the temperature was changed in the reverse direction, a transient increase of the frequency was noted. This transient response to the temperature change was the origin of the dependence of the frequency of directional changes on the swimming direction in the temperature gradient. Finally, the relation between the magnitude of the transient response and the rate of the temperature change was derived.     

Disparity of native oxyhemoglobin components isolated from Paramecium caudatum and Paramecium primaurelia

• 1.1. Native oxyhemoglobin components were isolated chromatographically from Paramecium caudatum and Paramecium primaurelia, and some properties of the isolated components were investigated.
• 2.2. P. caudatum was endowed with one homogeneous hemoglobin component, while the hemoglobin in P. primaurelia was resolved into three heterogeneous components being two main and one minor.
• 3.3. Spectral properties of the isolated hemoglobin components were quite similar to each other. The isolated components, however, were distinctly different in electrophoretic mobilities.
• 4.4. Molecular weight of the isolated hemoglobin components was estimated to be about 11,000.

     

Binding of Ca ions by Paramecium caudatum

Binding of ⁴⁵Ca by live Paramecium caudatum was determined under various external ionic conditions. It was found that calcium uptake was separable into at least two components, a rapid and a slow one. The rapid component was influenced by the presence of certain other ions in a manner which agrees with the law of mass action. It appears that an ion exchange system may be involved in a binding equilibrium established between Paramecium, Ca⁺⁺, and certain other ions. K⁺, Rb⁺, and Ba⁺⁺ in the equilibrium medium are among those ions which inhibit calcium uptake. It is proposed that liberation of Ca⁺⁺ from binding sites on Paramecium by an exchange reaction with competing ions is the first step in the mechanism of ciliary reversal in the response to external application of these ions.     

Serotypes and Immobilization Antigens in Paramecium caudatum*

SYNOPSIS. Some of the serotypes in Paramecium caudatum are described in this paper. Immobilization antigens of P. caudatum have been obtained by extracting paramecia in a dilute salt solution containing 15% alcohol. Immobilization antigen F from stock 162 has been isolated and has a sedimentation coefficient of 9.3 Svedbergs, diffusion coefficient of 2.3 × 10^-7 cm/sec, and molecular weight of approximately 340,000.     

Macronuclear Regeneration and Cell Division in Paramecium caudatum

SYNOPSIS. In Paramecium caudatum , occurrence of macronuclear regeneration is closely related to the time of feeding after conjugation. Macronuclear regeneration is induced with a high frequency when conjugating pairs are transferred into fresh culture medium. Feeding immediately after conjugation induces early cell division and 3 or more fissions occur without macronuclear division because of the inability of the macronuclear anlagen to divide. In the cells lacking normal macronuclear anlagen, old macronuclear fragments undergo regeneration and form vegetative macronuclei.     

Ciliary Membranes and Mating Substances in Paramecium caudatum

SYNOPSIS Cilia detached from mating reactive cells of Paramecium caudatum were fractionated for the purpose of identifying the structural component bearing mating substances. Purified axoneme fractions had no mating reactivity. The membrane fraction obtained by dialyzing against a solution of Tris-EDTA (0.1 m m EDTA, 1 m m Tris-HCI, pH 7.6) and 0.6 m KCI, and then by centrifuging over 40% (w/v) sucrose was strongly reactive. No mating reactivity was detected in the soluble fractions containing axonemal and matrix proteins. The results indicate that the mating substances in active from are localized only on the ciliary membranes.     

Isolation and characterization of the hemoglobin from Paramecium caudatum

• 1.1. The hemoglobin of Paramecium caudatum has been purified to a state of homogeniety on polyacrylamide disc electrophoresis.
• 2.2. The hemoglobin appears as a single molecular species with a mol. wt of 13,500 daltons as determined by SDS disc electrophoresis.
• 3.3. An isoelectric point of 4.27 was calculated from isoelectric focusing experiments using ampholines in a pH range of 3–6.
• 4.4. The amino acid composition is: lys₅, his₂, arg₃, asp₁₂, thr₁₁, ser₉, glu₁₆, pro₂, gly₁₈, ala₂₁, cys₀, val₁₂, met₂, ile₂, leu₈, tyr₂, phe₆.
• 5.5. The spectra of three ferrous and the ferric cyanmethemoglobin derivatives are presented.

     

Electrical properties of Paramecium caudatum: all-or-none electrogenesis

Summary Specimens of Paramecium immersed in solutions of CaCl₂ show graded electrogenesis in response to imposed transmembrane current. However, when BaCl₂ in a final concentration of 0.25 mM is added to a 1 mM CaCl₂ solution, an outward current pulse of 10^-10 amp or greater elicits an all-or-none transient reversal in membrane potential having a duration of about 40 msec. An increase of [Ba⁺⁺] results in (a) lower resting potential, (b) positive shift in critical firing level, (c) increased overshoot of the action potential, (d) decreased hyperpolarizing afterpotential, and (e) increased duration of the action potential (a.p.). If [Ca⁺⁺] is increased along with [Ba⁺⁺] so as to keep the ratio [Ba⁺⁺]/[Ca⁺⁺] constant, the same results are obtained except that the duration of the a. p. remains unaltered. Thus, effects a-d appear to be related to [Ba⁺⁺] and not to [Ca⁺⁺] or [Cl^-]. The degree of overshoot in 1 mM Ca is linearly related to log [Ba⁺⁺] with a slope of approximately 22 mv. With the ratio [Ba⁺⁺]/[Ca⁺⁺] constant, the slope closely approaches the ideal value of 29 mv. The evidence indicates that prolongation of the action potential is due to a delayed onset of Ba inactivation, and that this in turn is a function of surface-bound Ba. Other features of the action potential are absolute refractoriness during its rising and plateau phases, relative refractoriness lasting several seconds, and repetitive firing in response to steady current depolarization. The response is unaffected by TTX and TEA. Mn prolongs the action potential. Sr has an action similar to Ba, whereas the addition of K, Na, Rb, or Mg to the basic calcium medium is unaccompanied by all-or-none electrogenesis.On leave of absence from the Zoological Institute, Faculty of Science, University of Tokyo.Support came from National Science Foundation grant GB-5752x, U.S.P.H.S. grant NB-03664, and in part from Office of Naval Research grant Nonr 4785(00) administered by the Marine Biological Laboratory, Woods Hole.     

Mating-reactive membrane vesicles from cilia of Paramecium caudatum

Membrane vesicles with a high mating reactivity were obtained from cilia of Paramecium caudatum by treatment with a solution containing 2 M urea and 0.1 mM Na2-EDTA. All processes of conjugation were induced in cells of the complementary mating type by approximately 10 mug/ml proteins of the vesicles. Electron microscope observation showed that the membrane vesicles have a diameter of 100-150 nm. Electrophoretic analysis on SDS polyacrylamide gel revealed no significant difference in polypeptide patterns of the particles from the two complementary mating types.     

Detection of triclosan using 2-domain hemoglobin promoter of Daphnia magna

Triclosan has been widely used as an antimicrobial agent. However, triclosan was found to cause toxicity, including muscle contraction disturbances, carcinogenesis, and endocrine disorders. In addition, it was found to affect central nervous system function adversely and even have ototoxic effects. Conventional methods for detecting such triclosan can be performed easily. However, the conventional detection methods are inadequate in precisely reflecting the impact of toxic substances on stressed organisms. Therefore, a test model for the toxic environment at the molecular level through the organism is needed. From that point of view, Daphnia magna is being used as a ubiquitous model. D. magna has the advantages of easy cultivation, a short lifespan and high reproductive capacity, and high sensitivity to chemicals. Therefore, the protein expression pattern of D. magna that appear in response to chemicals can be utilized as biomarkers for detecting specific chemicals. In this study, we characterized the proteomic response of D. magna following triclosan exposure via two-dimensional (2D) gel electrophoresis. As a result, we confirmed that triclosan exposure completely suppressed D. magna 2-domain hemoglobin protein and evaluated this protein as a biomarker for triclosan detection. We constructed the HeLa cells in which the GFP gene was controlled by D. magna 2-domain hemoglobin promoter, which under normal conditions, expressed GFP, but upon triclosan exposure, suppressed GFP expression. Consequently, we consider that the HeLa cells containing the pBABE-HBF3-GFP plasmid developed in this study can be used as novel biomarkers for triclosan detection.     

An Inhibition and Kinetic Study of Acid Phosphatase in Paramecium caudatum and Paramecium tetraurelia1

ABSTRACT. Inhibition, inactivation, pH, and kinetic studies using both homogenates and purified lysosomal fractions of Paramecium caudalum and of P. tetraurelia were carried out to examine the lysosomal acid phosphatase (AcPase) and its relationship to p-nitrophenylphosphatase (pNPPase), glucose-6-phosphatase (G6Pase), and 5′-nucleotidase (AMPase). The results generally support the idea that Paramecium cells contain a distinct lysosomal AcPase with a broad substrate specificity. The hydrolysis of glucose-6-phosphate (G6P) and adenosine 5′-monophosphate (AMP) was shown to be due to this enzyme, suggesting that true G6Pase and AMPase may be lacking in these two species; however, some hydrolysis of AMP at pH 7.5 catalyzed by an unknown soluble enzyme distinct from alkaline phosphatase and Na⁺-K⁺-ATPase was observed. Since the hydrolysis of p-nitrophenylphosphate (pNPP) at acid pH was also shown to be due to AcPase alone, pNPPase could be used as a rapid assay for Paramecium AcPase. At an alkaline pH, however, this activity was catalyzed by an alkaline phosphatase located in the cytosol fraction. P. caudatum AcPase was shown to have kinetic properties similar to those of purified rat liver and human prostatic AcPase and to have relative substrate affinities in the order of G6P < β-glycerophosphate < pNPP < AMP. These different substrate affinities might account for the observed differences in the inhibition of the four lysosomal activities by NaF, L(+)-tartrate, and molybdate, all of which inhibited the hydrolysis of G6P, β-glycerophosphate, and pNPP competitively, but which exhibited a noncompetitive inhibition of a mixed type with the hydrolysis of AMP.     

Application of individual growth and population models of Daphnia pulex to Daphnia magna, Daphnia galeata and Bosmina longirostris

Daphnia models for individual growth and population dynamics have been developed in the manner of models developed by Gurney, McCauley, Andersen and others. All or most of the earlier models were parameterized for Daphnia pulex; we have used the D. pulex model as a baseline model for other species of Daphnia such as magna, galeata and also Bosmina longirostris. Because of the lack of ample data for D. magna, D. galeata and B. longirostris, some of the physiological data had to be relied on the other species whose data were available and in some case calibrated. We were able to produce reasonable results for individual growth as well as population dynamics under the controlled laboratory conditions. Most of the results were compared with the available laboratory data for population as well as growth. All the simulations have been done under high and low food concentrations. The animals are assumed to be feeding on green algae (Chlamydomonas reinhardtti) under the laboratory conditions of 18–20°C. The continuous growth until the end of the life was observed in smaller B. longirostris, whereas rapid growth in the beginning and slower after the start of the reproduction was observed in Daphnia species. The smaller species matured earlier than larger species. B. longirostris population sustained better than Daphnia species in medium food concentrations.     

Reproducibility of short-term and reproduction toxicity experiments with Daphnia magna and comparison of the sensitivity of Daphnia magna with Daphnia pulex and Daphnia cucullata in short-term experiments

Short-term toxicity tests with Daphnia magna are reasonably reproducible. The sensitivity of Daphnia pulex, Daphnia cucullata and Daphnia magna proved to be about the same in short-term tests with 15 different chemical compounds.Reproduction toxicity experiments with Daphnia magna can easily be carried out within three weeks. At least duplicate experiments are necessary to arrive at an approximate no-effect level.     

Macromolecular isoforms of Daphnia magna haemoglobin

The haemoglobin (Hb) of Daphnia magna acclimated to different oxygen conditions was sampled, and in its natively assembled state it was separated by chromatofocusing. The Hb isoforms were analysed for their subunit composition under denaturating conditions by two-dimensional gel electrophoresis. The Hb system is suggested to consist of three predominant Hb aggregates, which are characterised by a specific subunit composition and synthesised in response to different ambient oxygen conditions. In normoxia, a dominant Hb aggregate (DmHbI) with a pI of 4.4-4.6 was composed of subunits B, C, E, F and G. In severe hypoxia, a different dominant Hb isoform (DmHbIII) with a pI of 5.7-5.9 was composed of subunits A, B, C, D, E and F. Further analyses in moderate hypoxia provided evidence for a third Hb isoform (DmHbII) composed of subunits B, C, D, E and F. Sequence alignment and homology modelling of the tertiary structure of the D. magna Hb domains 1 and 2 revealed functionally relevant substitutions of amino acid residues at positions B10, E7 and E11, which determine the functional properties of D. magna haemoglobin in terms of haem contact, oxygen binding and affinity. Both domains are predicted to possess the common haemoglobin fold, but helices C and D are not properly formed, and helix G is interrupted by a short coil.