Yoshiomi Takagi

Purification and identification of a growth factor produced by Paramecium tetraurelia

We previously reported that the jumyo mutant of a cilate protozoan Paramecium tetraurelia excretes into the medium a factor which promotes its own cell division. Here, the factor was purified to electrophoretic homogeneity through a series of liquid chromatographic procedures. This substance is a protein with a molecular weight of 17,000 which at concentrations of 1 X 10(-9) M (17 ng/ml) or more results in the recovery of the cell division rate of the jumyo mutant to the level of the wild type. The factor is therefore considered to be a growth factor and was named Paramecium growth factor (ParGF). This is the first report of direct proof for the production of a growth factor in an organism other than vertebrates.

A Substance Secreted from Tetrahymena and Mammalian Sera Act as Mitogens on Paramecium tetraurelia

Abstract We previously isolated and purified Paramecium growth factor (ParGF) from a cell-free fluid of an early stationary mass culture of Paramecium tetraurelia (Tanabe et al., 1990). The mitogenic activity of the purified ParGF and of the crude sample (ca. a 100-fold concentrate obtained by ultrafiltration of cell-free fluid) has been assessed based on restoration of the fission rate of the jumyo mutant of P. tetraurelia in daily reisolation cultures. With this assay system, we found that crude samples of Tetrahymena pyriformis and T. thermophila showed mitogenic activity. This suggests that Tetrahymena cells secrete a mitogenic factor(s) like ParGF. To some extent, fetal bovine serum (FBS) and calf serum (CS) also acted as mitogens on the jumyo mutant. Of nine mammalian growth factors assayed for their mitogenic effects on the jumyo mutant, epidermal growth factor (EGF) and transforming growth factor &agr; (TGF&agr;) were slightly and occasionally effective. These results support the idea of actual use of similar kind of growth factors to control cell divisions from protozoa to mammals.

Factors Controlling the Length of Autogamy-Immaturity in Paramecium tetraurelia

Abstract Autogamy-immaturity is the period during which autogamy can not be induced by natural starvation; in Paramecium tetraurelia, autogamy first becomes induceable at about 7 fissions after the previous autogamy, and thereafter the percent of cells undergoing autogamy increases gradually to almost 100% at the clonal age of about 20 fissions and remains at 100% thereafter. The length of autogamy-immaturity (LAI), determined by plotting the percent of cells in autogamy versus the number of fissions, was found to be similar in two cultures grown at different fission rates at 25°C and 30°C. This indicates that paramecia count LAI by the number of fissions, not by the calendar time. LAI estimated from the peaks of percent autogamy through successive autogamous generations was also similar in two continuous cultures grown with different cycles of growth and starvation at 25°C and 30°C, indicating stability of LAI under ordinary laboratory conditions. However, LAI was affected by the cultural age of paramecia from which the new autogamous generation was derived: advanced cultural age shortened the LAI in the following generation.

Catalase is the Bacteria-Derived Detoxifying Substance against Paramecia-Killing Toxin in Wheat Grass Powder Infusion

Abstract Paramecium cells are usually cultured in a wheat grass powder infusion inoculated with Klebsiella pneumoniae. However, non-bacterized wheat grass powder infusion is toxic to paramecia, and bacteria-derived substance detoxifies the toxic substance. Here, the detoxifying substance from K. pneumoniae, which was found to be proteinaceous, was purified to homogeneity. The protein had an apparent molecular mass of about 200 kDa by gel filtration and 92 kDa by SDS-polyacrylamide gel electrophoresis. Although the amino acid sequence of the amino terminal region did not show a high sequence homology with any reported proteins, amino acid sequences of internal regions of the protein were nearly identical to catalase HPII from Escherichia coli. When the wheat grass powder infusion was treated at 25 °C for 1 h with commercially available catalase from bovine liver, the toxicity of the infusion against paramecia was completely abolished. The initial concentration of hydrogen peroxide in the wheat grass powder infusion was about 30 μM and was completely decomposed by the catalase treatment. Therefore, the toxic substance in the wheat grass powder infusion and the detoxifying substance from K. pneumoniae are considered as hydrogen peroxide and catalase, respectively.

Survival or extinction of Paramecium multimicronucleatum cultured with P. tetraurelia is associated with the ability or inability to form food vacuoles

Summary We reported previously that Paramecium multimicronucleatum (Pm) cultured with P. tetraurelia (Pt) in a closed environment became extinct at the stationary phase and that Pm and Pt coexisted when different stocks of Pm were used and when Pm and Pt were cultured in stigmasterol-containing medium. We proposed to explain the phenomena by survival substances with different thresholds for Pm and Pt; but the mechanism of how these substances work to change the fate has remained open. We here show that the fate of survival or extinction of Pm is associated with its ability or inability to form food vacuoles (FVs): FV formation of coexisting Pm was active and that of extinctive Pm was inactive. By addition of stigmasterol to the mixed culture in which Pm cells were dying out, their FV formation was activated in concomitance with their rescue from extinction; only the Pm cells with the ability to form FVs were rescued. We also show that Pm and Pt coexist in the medium to which cholesterol or ergosterol is added; these sterols retarded the growth of Pt cells so that Pm cells grew to a higher cell density, which helped them survive.

A Paramecium tetraurelia mutant that has long autogamy immaturity period and short clonal life span

We have isolated an unprecedented mutant of Paramecium tetraurelia that has a long immaturity period until autogamy. This mutant stock, d4-RK, screened for 0% autogamy at the age of 27 fissions, began to undergo autogamy around the age of 50 fissions in some clones and underwent autogamy scarcely even after the age of 100 fissions in others. d4-RK expressed its mutant phenotype at 25 degrees C, but resembled the wild-type phenotype at 32 degrees C. Genetic analyses indicated that a single recessive gene, named rie (remote immaturity exit), was responsible for the mutant phenotype. This is the first report to show a gene that elongates the time to sexual maturation in unicellular organisms. The clonal life span was shorter and fission rate was lower in the rie mutant than in the wild-type, both at 25 degrees C and 32 degrees C. Even in the fourth autogamous generation following the third backcross to the wild-type, the progeny with the elongated autogamy immaturity period still had a short clonal life span and low fission rate, while those with the wild-type phenotype in autogamy immaturity period showed the wild-type phenotypes in clonal life span and fission rate, too.

Abrupt Increase in UV Sensitivity at Late Log-Phase of Growth in Paramecium tetraurelia

Abstract. In this study, changes in UV sensitivity, a parameter of the clonal aging that has been studied in the daily reisolation culture, were examined in the logarithmically growing Paramecium culture. Cells in logarithmically growing cultures are thought to change the internal states under rapidly changing external conditions. In contrast, cells in daily reisolation cultures gradually change the internal states, the process being called clonal development and aging, under the external conditions that are kept almost constant. Cells were sampled at regular intervals, irradiated with UV, and examined for UV sensitivity assessed by the clonal survival. We found that log‐phase cells showed low sensitivity to UV until they reached 2,000–3,000 cells/ml, and beyond that cell density, abruptly became highly UV sensitive. The extent of this increase in UV sensitivity was similar to that between two age groups, 130 fissions of clonal age apart. When cells from a culture of 2,000–3,000 cells/ml were resuspended in culture medium at various cell densities, they changed to UV sensitive only when the cultures reached over ∼2,600 cells/ml. These results suggest that paramecia become UV sensitive in response to change in the nutrient level when cell density exceeds 2,000–3,000 cells/ml.

Conditions of survival and extinction of Paramecium multimicronucleatum cultured with P. tetraurelia in a closed environment

Summary With two species of Paramecium , P. multimicronucleatum (Pm) and P. tetraurelia (Pt), we explored in more detail the classical experiments of Gause from which the competitive exclusion principle was formulated. We found that when they were cultured together, Pm cells, but not Pt cells, became extinct after reaching their maximal density. The extinction of Pm in the mixed culture occurred not only in a semi-open system in which 1/10 of the culture was replaced daily with fresh culture medium, but also in a closed system to which no food was added. Pm cells survived, however, in the mixed culture when different stocks of Pm were used, when cultured in higher cell density of Pm, and when cultured in medium supplemented with stigmasterol or in medium prepared from Wheat Grass Powder of a new lot number. These results indicate that survival and extinction of Pm cultured with Pt are a physiological phenomenon rather than an ecological problem. We propose that the fate of Pm is affected by survival substances in the surrounding medium, which are composed largely of the remaining food and partly of the secreted growth factor, and by a threshold level for survival substances for Pm.

A new mutation in the timing of autogamy in Paramecium tetraurelia.

We have isolated a new type of Paramecium tetraurelia mutant, named rie-2, that has a long immaturity period until autogamy. We previously isolated such an autogamy mutant, designated rie-1. These two mutants had some additional common features such as dependence of the occurrence of autogamy on the temperature, involvement of a single recessive gene, lower fission rate and shorter clonal life span. However, rie-2 was considered a new type mutant distinguishable from rie-1 because of their different natures of temperature sensitivity. First, the temperature at which they resembled the wild-type phenotype was low (19 degrees C) in rie-2, although it was high (32 degrees C) in rie-1. Second, the clonal life span of rie-2 at 25 degrees C was similar to that of the wild-type, but it was extremely shorter at 32 degrees C than at 25 degrees C, although it was similarly shorter at both temperatures in rie-1. Third, the difference of the fission rate between mutant and wild-type was greater at 32 degrees C than at 25 degrees C in rie-2, although it was similar at both temperatures in rie-1. This report shows that a gene mutation to elongate the period until sexual maturation does not necessarily assure the long life span.

Age-associated survival and extinction in mixed cultures of Paramecium

When Paramecium tetraurelia (Pt) and P. multimicronucleatum (Pm) were cultured together, the outcome was either survival of Pt and extinction of Pm, or coexistence of Pt and Pm. We have shown that these fates were experimentally controllable by changing external culture conditions or by selecting stocks of Pm. However, we have so far failed to observe survival of Pm and extinction of Pt. We showed previously that the survival or extinction of Pm was associated with its ability or inability to form food vacuoles (FVs). An inability to form FVs is one of the characteristics of senescence in Paramecium. The extinction of Pm was observed only when stock CH312 of Pm was used, and this stock shows some senescent characteristics. Therefore, we studied the effects of clonal aging on the fate in mixed culture. Here, we report that all three possible fates including survival of Pm and extinction of Pt are controllable by changing the clonal age of Pt. Survival or extinction is also controllable between different age groups of the same species (Pt), indicating decisively that the phenomenon is not an inter-species problem. Cells without the ability to form FVs were found prior to the occurrence of extinction.