Membrane protein differences correlated with the development of mating competence in Tetrahymena thermophila

Initiation is the contact-independent phase of sexual conjugation which occurs when mature cells of Tetrahymena thermophila are shifted from growth medium to a low-salt starvation buffer. Immaturity, like high-salt starvation, restricts the ability of cells to conjugate; immature cells do not conjugate in either low- or high-salt buffers. Comparisons between sexually mature cells starved in initiation-restrictive and initiation-permissive buffers, and between immature and mature cells starved in an initiation-permissive buffer permitted the analysis of membrane protein expression correlated with mating competence. No polypeptides identified by lactoperoxidase-catalyzed iodination were found to be specific to mating-competent cells; however, several polypeptides not present in initiated cells were found to be common to the cell surfaces of immature and non-initiated cells which suggests that (1) initiation involves the removal of specific proteins from the cell surface, and (2) immaturity may be due to an inability to initiate.     

Cyclic AMP and its functional relationship in Tetrahymena: a comparison between phagocytosis and glucose uptake.

In Tetrahymena, an increase in the level of cAMP is accompanied by an increased phagocytotic rate, whereas increased sugar uptake is parallelled by a decreased cAMP level. The increase in cAMP level seems to be decisive with respect to phagocytosis as a basic phenomenon of life. In the action of epinephrine, however, some mechanism other than cAMP mediation may be involved. Depending on concentration, one hormone may provoke either an increase or a decrease in cAMP level, and this in turn triggers the corresponding function.     

Dual capacity for nutrient uptake in Tetrahymena. II. Role of the two systems in vitamin uptake.

Previously, we found that a mutant strain of Tetrahymena pyriformis without food vacuoles failed to grow unless the nutrient media were richly supplemented with vitamins and trace metals. Here we show that calcium folinate alone can replace the extra vitamin supplementation. The mutant requires approximately 90-fold higher concentration of folinate than the wild-type cells to give similar growth responses in a chemically defined medium. We infer that the food vacuole is an important route of uptake for this vitamin in the wild-type cells. We found no difference between mutant and wild-type cells in their requirements for nicotinic acid, pantothenic acid, riboflavin-monophosphate, and pyridoxal. We infer that an extravacuolar route contributes importantly to uptake of these 4 compounds.     

Modification of membrane lipids. Phenethyl alcohol-induced alteration of lipid composition in Tetrahymena membranes.

Tetrahymena pyriformis NT-I cells in the early-logarithmic phase were incubated with phenethyl alcohol (2-phenylethanol) and effects on the lipid composition were examined in various membranes. 1. There was a marked modification in phospholipid head, as well as fatty acyl group composition in pellicles, mitochondria and microsomes of the phenethyl alcohol-treated cells. Compared with membranes of the control cells, the membranes from phenethyl alcohol-treated cells were found to contain a higher level of phosphatidylcholine content with the compensating decrease in phosphatidylethanolamine, while 2-aminoethylphosphonolipid showed only a slight decrease in these membranes. The acyl group profile of membrane phospholipids in the presence of phenethyl alcohol was also modified so that a profound elevation of the content of polyunsaturated fatty acids, linoleic and gamma-linolenic acids. The major monounsaturate, palmitoleate decreased. Such lipid alteration is a reversible process, and therefore upon removal of phenethyl alcohol the modified lipid composition returned to normal. 2. By freeze-fracture electron microscopy in combination with temperature quenching, the outer alveolar membrane of the phenethyl alcohol-treated cell was observed to reveal less aggregation of intercalated-membrane particles, as compared with the control membrane. The quantitative analysis of the thermotropic lateral movement of membrane particles provided evidence that the membrane in the phenethyl alcohol-treated cell became more fluid. Such fluidizing effects may result from an increase in the acyl group unsaturation and also in the phosphatidylcholine content. 3. With regard to the mechanism responsible for the marked decrease in palmitoleate in membrane phospholipids, there was found a depressed conversion of the palmitate to palmitoleate in the phenethyl alcohol-treated cells. It was further suggested that the drug may have an inhibitory effect on the synthesis of palmitoyl-CoA desaturase involving the (16 : 0 leads to 16 : 1) conversion. Also, it was demonstrated that the increase in a precursor-product fashion of phosphatidylcholine with the corresponding decrease in phosphatidylethanolamine was not due to transformation of phosphatidylethanolamine to phosphatidylcholine through stepwise methylation.     

Imprinting, digoxin uptake and storage in Tetrahymena

Tetrahymenas after a single exposure to high concentration of digoxin, release glycoside into the culture medium even three days after removal of the drug. After nine or fifteen days, however, this effect subsides. Re-administration of digoxin causes the digoxin consumption (storage) of Tetrahymenas to increase. This observation indicates that the digoxin imprinting previously demonstrated in mammals occurs also in Tetrahymena. The experiments raise the possibility that also Tetrahymenas are capable of producing a digoxin-like substance detectable by radioimmunoassay.     

Population dynamics of Yersinia pseudotuberculosis in association with the infusorian Tetrahymena pyriformis

The decisive role of biotic factors in the preservation of the viability of Y. pseudotuberculosis is shown. The results of experiments suggest that protozoa may serve as reservoir in the spread of Y. pseudotuberculosis infection.     

Early prostate development and its association with late-life prostate disease

The development of the prostate is an emerging priority area for prostate biologists. Early changes in prostate development permanently alter prostate morphology and function and an understanding of the permanent nature of early events that may influence the onset of late-life disease is vital. Two of the inherent problems involve associating exposure in early life with outcome in late life or maturity and accounting for the influence of genetic, environmental, dietary or metabolic factors during the intervening period. Any one of these factors, alone or in combination, might lead to an explanation of the discrepancies found in the literature regarding the influence of early changes to the prostate in later life. Therefore, it is important to establish a causal link between the hormonal changes that occur during the fetal/neonatal period and that imprint the gland and the onset of late-life pathology. In order to achieve this goal, several technical challenges need to be overcome to permit the objective assessment of prostate branching morphogenesis. Stereological techniques now allow the quantification of several parameters of branching morphogenesis and the identification of specific early changes that are permanent and irreversible with a late-life outcome. This methodology provides the means to determine the action of a range of genes or hormone/growth factors that have been implicated in prostate development and disease. This study is supported by an NH&MRC program grant number 973218.     

Acetylcholinesterase and its association with lipid.

Human erythrocyte acetylcholinesterase preparations which vary in lipid content, from lipid-rich to lipid-poor, have been successfully prepared using deoxycholate. It was found that the lipid content of the enzyme decreased gradually as the deoxycholate concentration used in its preparation was increased. The binding of lipid to lipid-poor preparations of the enzyme has been investigated. It was found that the activity of such preparations was highly dependent on their phospholipid contents. Maximum specific activity was associated with a fixed phospholipid content. The lipid-poor enzyme was highly activated by addition of either endogenous(membrane) or exogenous lipid. Based on the data presented, it was concluded that acetylcholinesterase is a phospholipoprotein, and its activity is highly dependent on its phospholipid component.     

Constitutive and Induced Excretion of Polypeptides in Tetrahymena thermophila1

Polypeptides normally excreted to growth or starvation media were revealed using O'Farrell gel-electrophoresis and silver-staining. The major polypeptides detected in conditioned media were either constitutive, growth specific, starvation specific, or high-Tris specific. The majority of the excreted polypeptides could be released from the cells by a hypo-osomotic shock, possibly resulting in membrane leakage, but also mucocyst and/or plasma membrane-associated polypeptides were detected in the conditioned media.     

Polymorphisms in the hepatitis C virus core and its association with development of hepatocellular carcinoma

Little is known about the mechanisms underlying hepatocellular carcinoma (HCC). Some studies have focused on the role of HCV viral proteins in hepatocyte transformation. In this work we have compiled and analysed current articles regarding the impact of polymorphisms in the HCV core gene and protein on the development of HCC. An exhaustive search for full-text articles until November 2016 in PubMed database was performed using the MeSH keywords: ‘hepatitis C’, ‘polymorphisms’, ‘core’, ‘hepatocellular cancer’ and ‘hepatocarcinogenesis’. Nineteen full-text articles published between 2000 and 2016 were considered. Different articles associate not only the HCC development with polymorphisms at residues 70 and 91 in the core protein, but more with mortality and treatment response. Also, different polymorphisms were found in core and other viral proteins related to HCC development. Eleven articles reported that HCC development is significantly associated with Gln/His70, four associated it with Leu91 and two more associated it with both markers together. Additional studies are necessary, including those in different types of populations worldwide, to validate the possibility of the usability and influence in chronically HCV-infected patients as well as to observe their interaction with other risk factors or prognosis and genetic markers of the host.     

Kinetic properties of dynein ATPase from Tetrahymena pyriformis. The initial phosphate burst of dynein ATPase and its interaction with ATP analogs.

1. Dynein was extracted with 0.5 M KCl from Tetrahymena axonemes. SDS-gel electrophoresis of the extract indicated that about 50% of the extracted protein had a molecular weight of about 3.5 X 10(5), and that 90% of the proteins with this weight had been extracted. 2. The ATPase [EC 3.6.1.3] reaction of the KCl-extracted dynein fraction was enhanced by 60-80% by addition of the outer doublet fraction. It showed an initial burst of Pi liberation of about 1 mol per mol of proteins with a molecular weight of 3.5 X 10(5). 3. We examined the interaction of the dynein-tubulin system from Tetrahymena cilia with ten ATP analogs [2'-dATP, 3'-dATP, epsilonATP, FTP, 8-NH(CH3)-ATP, 8,3'-S-cyclo-ATP, 8-Br-ATP, 8-OCH3-ATP, 8-SCH3-ATP, and AMPPNP]. Among them, 2'-dATP and 3'-dATP were good substrates for dynein ATPase, as they induced the dissociation of dynein arms from the B-tubule of outer doublets, the sliding movement between outer doublets, and the bending movement of axonemes. The other analogs did not induce the dissociation or the sliding movement. 4. Among the ATP analogs tested, only 2'-dATP and 3'-dATP induced the reorientation of cilia on the Triton model of Tetrahymena; the reorientation rates were smaller than that induced by ATP.     

DNA elimination and its relation to quantities in the macronucleus of Tetrahymena.

The macronucleus of Tetrahymena contains a large number of DNA molecules of subchromosomal size. They belong to about 270 species each one occurring at an average number of 45 copies. Macronuclei divide unequally and nothing is known of segregation control. This and the elimination and degradation of DNA during macronuclear amitosis make the clonal stability of macronuclei a problem of qualitative and quantitative control on a subchromosomal level. We studied the contribution of DNA elimination to the quantitative composition of the macronucleus cytophotometrically in single cells of different strains. This was done under standard conditions and under conditions known to influence the amount of macronuclear DNA. The following results were found: Elimination of DNA occurs at almost every division. The size of the elimination body is highly variable but still positively correlated with the macronuclear DNA content. In T. thermophila the amount of eliminated DNA is 2.5% of the G2 content and is not dependent on the growth state. It varies with species, amounting to as much as 8% in T. pigmentosa. During conditions which increase the macronuclear DNA content, very little DNA is eliminated. On the other hand, large amounts are eliminated under other conditions causing the macronuclear DNA content to decrease. DNA to be eliminated at division is synthesized at the same time as bulk DNA. We developed a computer program which helps us study the effects of DNA elimination and unequal divisions upon the copy numbers of subchromosomal DNA classes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gametic differentiation in Chlamydomonas reinhardi: cell cycle dependency and rates in attainment of mating competency

Withdrawal of a utilizable nitrogen source during mid G₁ of the cell cycle induces gametic differentiation in synchronously grown vegetative cultures of Chlamydomonas reinhardi. Cell division accompanies gametic differentiation in such cultures, and the ability of mid G₁ vegetative cells to form gametes is matched by their ability to undergo a round of cell division after nitrogen withdrawal. Synchronously grown cultures require up to 19 hr in nitrogen-free medium to complete a round of division and to form mating-competent cells. Asynchronously grown liquid cultures require less time after nitrogen withdrawal (generally 5–8 hr) to achieve mating competency. In these cultures cell division did not necessarily accompany gametic differentiation since gametic differentiation took place in induced cultures at high cell concentrations which prevented cell division. Maximum mating competency was achieved in less than 2 hr after induction of vegetative cells grown on agar plates. Little cell division was observed during that short induction interval. The relationship between the attainment of mating competency (gametogenesis) and other physiological events resulting from nitrogen withdrawal is discussed.     

Development of the ciliature of Tetrahymena thermophila: I. Temporal coordination with oral development

The number of basal bodies and cilia along pole-to-pole ciliary rows was enumerated in Tetrahymena thermophila cells sampled during the rapid-exponential phase of culture growth in three different media that supported generation times ranging from 2 to 4 hr. The time required for oral development was nearly constant in the three media, and thus most of the differences in generation time were accounted for by differences in the interval prior to the onset of oral development (stage 0), which ranged from 50% of the generation time in the “poorest” medium to 20% in the “richest.” There was very little increase in number of basal bodies and of cilia along ciliary rows during stage 0, irrespective of the duration of this stage. The bulk of the increase took place during oral development, following a time course suggestive of coordination wth oral development. The same temporal pattern of increase was found in several ciliary rows, although the proportion of basal bodies that were ciliated differed among rows. There is no simple relationship between the number of basal bodies along ciliary rows and cell length, surface area, or volume. However, a large and constant proportion of the total division-to-division cell growth took place during the interval prior to the onset of oral development, suggesting that an ensemble of developmental events, including oral development and an associated activation of the remainder of the cell surface, may be triggered by attainment of a threshold cell size.     

Modeling the Individual Growth of Tetrahymena Sp. and its Population Consequences

ABSTRACT. Three types of mathematical growth models are presented to describe the individual growth of the ciliate Tetrahymena sp. feeding on the bacterium Pseudomonas fluorescens . Both organisms were isolated from a domestic waste-water treatment plant. Growth of individual ciliates and the consequences for the whole population are considered. Experimental data, obtained by following the individual ciliate during its lifespan from cell division to cell division, are used for parameter estimations. Differences between growth models for individuals turn out to have little effect on the specific population growth rate and the mean cell volume. In case of exponential growth of individuals the unstructured and structured population models are equivalent, even in time-variant environments. This knowledge can be applied in the stability analysis of food chains or forced systems. The results obtained facilitates quantification of protozoa biomass as a function of bacterial biomass in chemostats. More specifically, it highlights the dynamic behaviour of bacteria and protozoa in waste-water treatment plants.     

Reciprocal induction of cell division by cells of complementary mating types in Tetrahymena

Chick embryo fibroblasts in monolayer culture were synchronized by contact inhibition and serum starvation. Nuclear DNA isolated from the [³H] thymidine pulse-labelled cells throughout the period of DNA synthesis (S phase) was analysed by hydroxylapatite chromatography after renaturation at different C₀t values. It is shown that repeated sequences having different frequencies of reassociation, replicate differently throughout the S period. In order to study the distribution of the repeated sequences, DNA isolated during the S period was fractionated according to its buoyant density. It is shown that only some of the highly reiterated sequences which are included in the high buoyant density DNA fractions, replicate equally well during the early and the late S periods. By contrast, reiterated sequences of the low buoyant density DNA fractions replicate mainly during the late S period.