Inhibition of conjugation in Tetrahymena pyriformis by cerulenin. Possible requirement for de novo lipid synthesis.

Conjugation in Tetrahymena pyriformis is induced by the mixing of two starved complementary mating types. Addition of the antibiotic cerulenin, a specific inhibitor of de novo lipid synthesis, upon mixing of the mating types inhibited the conjugation process. The inhibition of conjugation was found to be reversible upon washing the cells. Cerulenin inhibited [14C]acetate incorporation into the lipid fraction of the cells, while it did not affect the incorporation of [3H]leucine into proteins. Analysis of the fatty acid composition of the whole cells revealed that during conjugation the ratio of saturated to unsaturated fatty acids is markedly changed. While the ratio of saturated:unsaturated fatty acids is 0.30 in unconjugated cells, it reached a value of 0.45 in conjugated cells.     

Inhibition of DNA synthesis does not influence the H1 histone synthesis in Tetrahymena pyriformis

In the protozoan Tetrahymena pyriformis the DNA synthesis is stopped immediately and completely after addition of one of the two DNA synthesis inhibitors methotrexate + uridine and hydroxyurea to a cell suspension. However, the present experiments show, that the accumulation of labeled H1 histone in the inhibited cells is almost totally unaffected for more than two-thirds of a cell cycle after addition of either inhibitor.     

Inhibition by cerulenin of lipid synthesis in Crambe abyssinica tissues

Cerulenin is a potent inhibitor of fatty acid synthesis from ¹⁴C-labelled acetate in leaves and developing seeds of Crambe abyssinica. The antibiotic is equally inhibitory on the elongation of [1-¹⁴C]-oleic acid to erucie acid which is the major fatty acid of the seed. There is no significant inhibition of fatty acid desaturation in either tissue. Acylation of lipids is not a primary target of cerulenin's action.     

Characterization of peroxisomes in Tetrahymena pyriformis

Peroxisomes from Tetrahymena pyriformis contained catalase, d-amino acid oxidase, cyanide-insensitive fatty acyl-CoA oxidizing system, carnitine acetyltransferase, isocitrate lyase, leucine:glyoxylate aminotransferase and phenylalanine:glyoxylate aminotransferase. These activities, except carnitine acetyltransferase, were found at the highest levels in the light mitochondrial fraction, whereas the highest activity of carnitine acetyltransferase was found in the micotchondrial fraction. Sucrose density gradient centrifugation showed that the density of peroxisomes was approx. 1.228 g/ml and that of mitochondria was approx. 1.213 g/ml. When the light mitochondrial fraction was treated with deoxycholate or by freeze-thawing, most of the activities of catalase and isocitrate lyase were solubilized, whereas about half of the original activity of aminotransferase remained in the pellet fraction. Addition of fatty acid and clofibrate increased the activities of the cyanide-insensitive fatty acyl-CoA oxidizing system and isocitrate lyase in the peroxisomes. The activity of catalase was slightly increased by glucose and clofibrate; leucine:glyoxylate aminotransferase activity was significantly increased by clofibrate treatment.     

Inhibition of conjugation and cell division in Tetrahymena pyriformis by tunicamycin: A possible requirement of glycoprotein synthesis for induction of conjugation

Tunicamycin, a glucosamine-containing antibiotic inhibited the conjugation process of Tetrahymena pyriformis. Sexual pairing was prevented completely when 1.5 μg/ml of tunicamycin was added to a mixture of the two mating types. Tunicamycin caused preferential inhibition of glycoprotein synthesis in Tetrahymena pyriformis. At 1.5 μg/ml and 6 μg/ml tunicamycin inhibited by 40% and 60% respectively [³H]-glucosamine incorporation into material precipitated by ethanol, while it did not affect [¹⁴C]-leucine incorporation. Cell division was also inhibited when the drug was added either to the regular growth medium or to the starvation medium.     

The determination of available lysine in barley using Tetrahymena pyriformis W.

Attempts to use the microbiological procedure based on growth of Tetrahymena for the assay of available lysine in barley led to several technical problems. These were mainly due to interference by carbohydrate with the measurement of the growth response of the organism, either by cell counting or by optical density.The assay was modified to suit the measurement of available lysine in barley grains. Fine grinding of samples, predigestion with papain and selection of appropriate N-concentrations in the medium were found to be key factors in adapting the technique for barley.The availability of lysine in barley, as assessed by the modified technique was found to range from 62 to 73%. Heating and micronization of barley grains reduced availability to 54 and 51%, respectively.     

Factors influencing the pattern of dopamine secretion in Tetrahymena pyriformis

Dopamine production and secretion by the unicellular eukaryote Tetrahymena pyriformis were examined through the use of high performance liquid chromatography (HPLC) with electrochemical detection and through labeling studies with radioactive precursors. Growing cultures maintained a steady state intracellular level of 1.6 ± 0.3 pmol dopamine/10⁶ cells while secreting dopamine into the medium at a rate of 0.2–0.3 pmol/10⁶ cells per min. Incorporation of [¹⁴C]tyrosine and l-[³H]dihydroxyphenylalanine (DOPA) into dopamine was most successful in a basal medium (1.3 mM Tris-HCl, 1 mM citric acid, and 1 mM Ca(OH)₂, (pH 6.5)). A rapid conversion of added l-[³H]DOPA into dopamine confirmed the dynamic pattern of dopamine synthesis and secretion first indicated by the quantitative chromatographic analyses. The intracellular concentration of dopamine dropped sharply after cells were resuspended in the basal medium at 10⁶ cells/ml, so that by approx. 1 h after resuspension, dopamine dropped below the level detectable by HPLC (0.15 pmol/10⁶ cells). Under these conditions, dopamine secretion continued at a high rate for some time, finally leading to a maximal extracellular concentration of 8.71 ± 1.73 pmol/ml by 1 h. At this concentration, the rate of secretion appears to match that of degradation. Pulse chase experiments confirmed the rapid 3urnover of intracellular dopamine. Approx. 90% of [³H]dopamine and l-[³H]DOPA disappeared from l-[³H]DOPA-prelabeled cells during a 5 min chase, with approx. 50% of this being recovered as [³H]dopamine in the cells' medium. Dopamine secretion could be increased by nearly 100-fold by adding high levels (15 nmol/ml) of l-DOPA to the medium. In contrast, NSD-1015, a potent inhibitor of dopamine synthesis, completely blocked dopamine production. 0.15 mM dibucaine and 0.02 mM reserpine reduced dopamine secretion by approx. 65% over a 25-min incubation, but 5 mM EGTA had no noticeable effect.     

Purification and characterization of extracellular acid phosphatase of Tetrahymena pyriformis

An extracellular acid phosphatase secreted into the medium during growth of Tetrahymena pryiformis strain W was purified about 900-fold by (NH₄)₂SO₄ precipitation, gel filtration and ion exchange chromatography. The purified acid phosphatase was homogenous as judged by polycrylamide gel electrophoresis and was found to be a glycoprotein. Its carbohydrate content was about 10% of the total protein content. The native enzyme has a molecular weight of 120 000 as determined by gel filtration and 61 000 as determined by sodium dodecyl sulfate-polycrylamide gel electrophoresis. The acid phosphatase thus appears to consist of two subunits of equal size. The amino acid analysis revealed a relatively high content of asparic acid, glutamic acid and leucine. The purified acid phosphatase from Tetrahymena had a rather broad substrate specificity; it hydrolyzed organic phosphates, nucleotide phosphates and hexose phosphates, but had no diesterase activity. The K_m values determined with p-nitrophenyl phosphate, adenosine 5′-phosphate and glucose 6-phosphate were 3.1·10^?4 M, 3.9·10^?4 M and 1.6·10^?3 M, respectively. The optima pH for hydrolysis of three substrates were similar (pH 4.6). Hg²⁺ and Fe³⁺ at 5 mM were inhibitory for the purified acid phosphatase, and fluoride, L-(+)-tartaric acid and molybdate also inhibited its cavity at low concentrations. The enzyme was competitively inhibited by NaF (K_i=5.6·10^?4 M) and by L-(+)-tartaric acid (K_i = 8.5·10^?5 M), while it was inhibited noncompetitively by molybdate K_i = 5.0·10^?6 M). The extracellular acid phosphatase purified from Tetrahymena was indistinguishable from the intracellular enzyme in optimum pH, K_m, thermal stability and inhibition by NaF.     

Mitochondrial respiratory chain of Tetrahymena pyriformis. The thermodynamic and spectral properties

The mitochondria isolated from the ciliate protozoon Tetrahymena pyriformis carry an oxidative phosphorylation with P/O ratio of 2 for succinate oxidation and P/O ratio of 3 for the oxidation of the NAD-linked substrates. The respiration is more than 90% inhibited with 1 mM cyanide while antimycin A and rotenone inhibit at concentrations of 1000-fold higher than those effective in mammalian mitochondria.Using a combination of spectral studies and potentiometric titrations, the components of the respiratory chain were identified and characterized with respect to the values of their half-reduction potentials. In the cytochrome bc₁ region of the chain a cytochrome c was present with an E_m7.2 of 0.225 V and two components with absorption maxima at 560 nm and the half-reduction potential values of ?0.065 and ?0.15 V at pH 7.2. The cytochrome with the more positive half-reduction potential was identified as the analogue of the cytochrome(s) b present in mitochondria of higher organisms, while the cytochrome with the more negative half-reduction potential was tentatively identified as cytochrome o. In addition ubiquinone was present at a concentration of approx. 4 nmol per mg mitochondrial protein.In the spectral region where cytochromes a absorb at least three cytochromes were found. A cytochrome with an absorption maximum at 593 nm and a midpoint potential of ?0.085 V at pH 7.2 was identified as cytochrome a₁. The absorption change at 615–640 nm, attributed usually to cytochrome a₂ was resolved into two components with E_m7.2 values of 0.245 and 0.345 V. It is concluded that the terminal oxidase in Tetrahymena pyriformis mitochondria is cytochrome a₂ which in its two-component structure resembles cytochrome aa₃.     

Reconstruction of oral cavity of Tetrahymena pyriformis utilizing high voltage electron microscopy

Reconstruction of the oral cavity from 17 0.5 μm thick serial sections observed with a high voltage electron microscope (JEM-1000, operated at 1000 kV) has enabled us to describe the in situ shape of the cavity, the orientation of the membranelles, oral ribs, cytostomal lip and forming food vacuole of Tetrahymena pyriformis. The study also showed that many different sets of microtubules encircle the oral cavity forming an interwoven, basket-like structure around the cavity thus providing it with considerable structural rigidity. By correlating results obtained from the reconstruction with results obtained from scanning electron microscopy and freeze-fracturing we have been able to elucidate the probable mechanism of how food particles are propelled into and through the oral cavity.     

High performance liquid chromatographic analysis of catecholamines in growing and non-growing Tetrahymena pyriformis

Tetrahymena pyriformis, strains NT-1 and W, harvested in logarithmic (growing) and stationary (non-growing) phases, were found by high-performance liquid chromatography to contain considerable quantities of dopamine. In addition, small amounts of epinephrine and norepinephrine were detected. Logarithmic-phase strain NT-1 cells contained 249±44 pg dopamine/10⁶ cells compared to 477±42 pg/10⁶ cells for logarithmic-phase strain W cells for logarithmic-phase strain W cells. The dopamine content of stationary-phase cells was approximately half the value of the logarithmic-phase cells. There was a significant amount of dopamine in the growth medium from stationary-phase cultures and, to a lesser extent, logarithmic-phase cells.     

Purification and partial characterization of cytochrome b5 from Tetrahymena pyriformis

With the use of detergents and successive column chromatographies, Tetrahymena b-type cytochrome was purified from microsomes to a specific content of 36.0 nmol per mg of protein. The purified form showed a single band on SDS-polyacrylamide gel with molecular weight of 22,000. The spectral properties of the reduced b-type cytochrome, the α-peak of which is situated at 560 nm and asymmetric with a shoulder at 556 nm, was different from that of rat liver microsomal cytochrome b₅. However, it was reducible by NADH in the presence of NADH-cytochrome b₅ reductase purified from rat liver microsomes.The results indicated that the microsomal b-type cytochrome should be designated as cytochrome b₅ of a ciliated protozoan, Tetrahymena pyriformis.     

Inhibition of conjugation in Tetrahymena pyriformis by ConA : Localization of ConA-binding sites

Two patterns of ConA binding to starved mating types of Tetrahymena pyriformis were observed depending on its time of addition. When ConA was added upon mixing of the mating types, at zero time of conjugation, it was first bound to the oral region and subsequently was taken into intracellular vacuoles. When it was added to conjugants, it was specifically bound as a ring around the conjugation area. The ability of the cells to form vacuoles, assayed by addition of carmine particles, declined prior to pair formation. The relationship between the above phenomena and the ability of ConA to inhibit conjugation is discussed.     

Inhibition of 6-methylsalicyclic acid synthesis by the antibiotic cerulenin.

Cerulenin, a specific inhibitor of beta-ketoacyl-(acyl carrier protein) synthetase [EC 2.3.1.41] and 3-hydroxy-3-methylglutaryl-CoA synthetase [EC 4.1.3.5], was studied to determine whether it inhibits 6-methylsalicylic acid synthesis, in which so-called "polyketide" formation, a condensation step similar to that in fatty acid synthesis, is involved. In fact, 100 mug/ml (4.5 X 10(-4) M) of cerulenin inhibited 60% of 6-methylsalicylic acid synthetase activity and 68% of fatty acid synthetase activity of Penicillium urticae.     

Inhibition of polyketide synthesis in Alternaria alternata by the fatty acid synthesis inhibitor cerulenin.

The fatty acid synthase inhibitor cerulenin (50 to 100 micrograms/ml) inhibited production of the polyketide mycotoxins alternariol (AOH) and alternariol monomethyl ether (AME) by the mold Alternaria alternata. The results suggested that AOH synthesis was inhibited by a direct mechanism by cerulenin, whereas production of AME was probably limited by a shortage of the precursor AOH.