RNA was isolated from Tetrahymena pyriformis GL and using human histidine decarboxylase (HDC) gene primers, the RT-PCR product was sequenced. A fraction containing 207 base pairs was compared to the published sequences of prokaryotic and mammalian (rat, mouse and human) HDC cDNA (exons). The HDC-cDNA fraction of Tetrahymena was similar to the mammalian cDNA-s and it was completely different from the prokaryotic HDC-gene. The results indicate the presence of a mammalian-like HDC-gene already in a unicellular eukaryote organism and demonstrates also that the divergence of the prokaryotic–eukaryotic common gene took place already at this low evolutionary level.

Proline-glycine, proline-leucine and proline-valine dipeptides and their retro variants were used in the experiments to study the effects of pretreatment (imprinting) in Tetrahymena, by investigating fluorescein isothiocyanate (FITC)-conjugated peptide binding. The protozoan organism could differentiate between the proline-dipeptides containing different partner amino-acids and between the dipeptides having the amino acids in reversed positions. The effect of imprinting was positive or negative and this was dependent on the type of the partner amino acid and on its position. Pro-Gly and Pro-Leu induced positive imprinting (elevated FITC-dipeptide binding) and Pro-Val induced negative imprinting (decrease of FITC-peptide binding). There was positive imprinting induction in two cases for the retro FITC-peptide and in one case for the FITC-conjugate of the imprinter peptide itself. The highest positive imprinting (almost 60% increase) was induced by Pro-Gly for FITC-Gly-Pro. Considering earlier—chemotaxis—experiments, the results of the present—binding—studies run parallel with the physiological effects. The experiments call attention to the sharp differentiating ability of small peptides at a unicellular level, that could have some role in the selection of molecules for hormone formation, during evolution.

Mitochondrial dehydrogenase activity was measured in seven taxa of Tetrahymena (T. pyriformis G1, T. hegewishi, T. malaccensis, T. pigmentosa, T. shapiro, T. thermophila CU-399, T. thermophila MS-1). Enzyme activity was different in the taxa investigated. Insulin reduced enzyme activity in six of the seven taxa studied. The duration of activity reduction was relatively long (5–10 min.) in most of the cases, and in T. hegewishi this lasted up to the end of the measurements (30 min.). There was no interrelation between the basic dehydrogenase activity of the taxon and the effect of insulin. There was also no correlation between the degree of relationship (of the taxa) and the dehydrogenase profile after insulin treatment.

Chemotactic properties of amino acids (L-alanine, glycine and L-lysine) and their oligopeptides (10 −6 M) and binding sites to these ligands were investigated in two unicellular models, the heterotrophic Tetrahymena pyriformis and the auxotrophic Dunaliella salina . Chemotaxis of Dunaliella induced by simple amino acids and their derivatives demonstrated that binding sites (receptors) for food molecules are not only present in the membrane but are also able to induce their basic physiological response. In Tetrahymena , substances with special molecular structure and properties (polar, hydrophilic character of the signal peptide chain)-5-L-Lys, 5-Glywere required for chemoattraction, other peptides tested, lacking the required structure, were repellent. Divergences in chemotaxis and binding assays of both species suggest that trends of functional and binding parameters do not run parallel at this level of evolution.

L-alanine and its peptides (L-Ala-2–6) do not attract or repulse Tetrahymena in a 10 −8 M concentration. In 10 −10 M concentration there is a consistent repellent effect. Twenty four hours after L-alanine or L-alanine-peptides' pretreatment (imprinting) the progeny generation of the cells react differently to the same materials. L-Alanine, L-alanine penta- and hexapeptide in both concentrations are chemoattractant, while L-alanine tetrapeptide is repellent. L-Alanine dipeptide is inert in 10 −10 M and repellent at 10 −8 M concentrations, while L-alanine tripeptide is strongly repellent at 10 −10 M and attractant at 10 −8 M concentrations. This means, that the first encounter (imprinting) with an exogeneous amino acid or peptide is decisive to the later reaction of the protozoan cell. The chain length is important in the imprinting, however the reaction is not consistent. The experiments call the attention to the significance of imprinting in the receptor and hormone evolution.

Gold-labeled insulin is bound first of all to the cilia of the oral field of Tetrahymena. A primary treatment (hormonal imprinting) with insulin increases the binding capacity even after 24h and makes it more sensitive for appearance a week later, within a minute of giving insulin-gold. The food vacuoles contain insulin-gold in pretreated cells or without pretreatment as well, though in imprinted situations the label can be found in pinocytotic vesicles at the bases of cilia in the oral field. Altogether, a functional difference can be observed between the cilia of the oral and non-oral surfaces of Tetrahymena and hormonal imprinting has a specifying effect on the binding of labeled hormone.

The unicellular tetrahymena contains inositol phospholipids (PI, PIP, PIP 2 ) and GPIs. Treatment with 10 −5 M insulin decreases the total 3 H-inositol incorporation and incorporation into PI. 24 h after 10 −6 M insulin treatment there is an elevation of these parameters. Second treatment with 10 −6 M insulin doubles and 10 −5 M decreases these levels. This means that the effect on phosphoinositide turnover by insulin in Tetrahymena is rather concentration dependent. Inositol incorporation into GPIs is also influenced by insulin.

The unicellular Tetrahymena possess hormone receptors in the nuclear envelope similarly to higher rank animals. These receptors bind insulin and their specificity is detectable by monoclonal antibodies developed to insulin. The hormonal (insulin) pretreatment (imprinting) of the cell did not alter the binding capacity of the nuclear membrane, demonstrated by antibody-technique. The specific binding characteristics of the plasma membrane was demonstrated and this was significantly increased following imprinting. In the nucleus of Tetrahymena presence of insulin was not detected by immunocytochemical method.

Tritiated diazepam accumulates mainly in the mitochondria of the unicellular Tetrahymena. This is the case in both a single (the first encounter) and a repeated (one day or a week after the first) administration of the drug. When imprinting of Tetrahymena by diazepam (the first encounter) is followed a week later by the administration of the labelled drug, the membranes of the vesicles, too, show the appearance of label. Regarding the studies presented here, the unicellular Tetrahymena also contain diazepam receptors in the mitochondria as suggested for cells of higher rank animals.

Histamine increased specifically the phagocytic activity of the unicellular Tetrahymena, whereas insulin had no influence on it. Insulin antagonized the phagocytosis stimulating action of histamine after simultaneous exposure and after preexposure two days earlier as well, although in the latter case to a lesser degree. Double exposure to a combination of histamine + insulin didn't influence the phagocytic activity at all, demonstrating the histamine antagonizing effect of insulin in this model.

Insulin and its A and B chain increased the quantity of intracellular PAS-positive material (glycogen) in tetrahymena, whereas the combined A+B chains decreased it. Imprinting—previous interaction—with insulin, its A and B chains in themselves and with the A+B chain increased the hormone binding capacity of tetrahymena, but the functional effect of imprinting (storage or breakdown of glycogen) showed a different tendency with insulin and A+B chain on the one hand, and A chain and B chain on the other. Since the imprinting potential of a molecule promotes the induction of receptor formation, the fact remains that both component chains of insulin were able to act as potential imprinters, although the A chain was superior to the B chain in this respect throughout, and combined treatment with the A+B chain ultimately induced the formation of a similar binding site as insulin itself.

The cells of the NIH 3T3 fibroblast line responded to primary interaction with insulin by a positive imprinting, i.e. by an increased binding capacity for the hormone on re-exposure. Positive imprinting, although to a lesser degree, was also induced by thyrotropin. However, oncogenic transformation by polyoma virus oncogens resulted in decreased imprinting in both the middle-T-antigen (MT3) and small-T-antigen-expressing (N4) cells.

Studies based on treatment with antibodies to thyrotropic hormone, luteotropic hormone, growth hormone or adrenocorticotropic hormone have shown that although the unicellular Tetrahymena does not possess sui generis receptors to all polypeptide hormones, such binding structures may arise, or become established in the membrane of the unicellular Tetrahymena in the presence of exogenous hormone. The Tetrahymena subjected to hormonal imprinting still contained an increased amount of hormone after six generation changes, which suggested that either hormone production had been induced by treatment, or the internalized hormone had not been degraded intracellularly. Thus the role of hormonal imprinting in receptor formation has also been substantiated by the immunocytochemical approach used in the present study.

Of the L and D configurations of four amino acids (phenylalanine, valine, tryptophan, tyrosine) tested for influence on the growth rate of Tetrahymena, only L-tyrosine was able to induce imprinting in Tetrahymena pyriformis Zeuthen. D-valine stimulated the division of T.pyriformis NT-1, but failed to induce imprinting. The experiments have substantiated the selectivity of the amino acid receptors of Y.pyriformis, and the extraordinary imprinting potential of tyrosine as well, as judged by its influence on the growth rate.

Certain non-hormone oligopeptides have a greater imprinting effect on Tetrahymena than others. The imprinting potential is unrelated to the length of the peptide chain, but seems greatly dependent on the amino acid sequence. The direct growth-stimulant action developed by the peptides at the first interaction is unrelated to their imprinting effect.