L. Köhidai

Rapid effects of insulin on cyclic GMP location in an intact protozoan

We studied rapid changes in location of cyclic GMP inTetrahymena pyriformis. Insulin caused cGMP localization in cilia and near the plasma membrane (0.5–1 min). Later (1–5 min) cGMP localization was diffuse in cytoplasm with perinuclear accentuation. Inactive insulin analogs did not elicit these changes.

Prolonged elevation of insulin content in the unicellular tetrahymena after insulin treatment: Induction of insulin production or storage?

In the unicellular organism, Tetrahymena, the first encounter with an exogeneously given hormone results in hormonal imprinting. This causes an increase of the binding capacity of receptors and the production of the appropriate hormone in the progeny generations of the treated cell. In the present experiments the quantity (using radioimmunoassay) and localization (using confocal laser scanning microscopy) of the immunologically insulin‐like material (hereafter insulin) were studied for 10 days after 4 h or 24 h 10−6 m insulin treatment (hormonal imprinting). Forty‐eight hours after both insulin treatments a high quantity of insulin was present in the cells. This value was also significantly increased after 96 h. After 8 days the difference to the control was significant only in the 24 h treated group. Confocal microscopy (using antibody to pig insulin) localized insulin in the cell body. The oral field contained extremely high quantities of the endogeneous hormone. Insulin treatment (after 48 and 96 h) caused an elevation of insulin content in general, and specific accumulation in the posterior sections of the cell, around the nucleus and in the periphery were observed. Ten days after both treatments only the peripheral region of the cell body and the ciliary row contained more insulin than the control. This means that after insulin treatment the quantity of insulin increases for a lengthy time period which is followed by the expression of insulin in the peripheral region. Insulin contained by Tetrahymena 48 h after imprinting stimulated glucose uptake of rat diaphragm. Copyright

Effects of the mammalian vasoconstrictor peptide, endothelin-1, on Tetrahymena pyriformis GL, and the immunocytological detection of endogenous endothelin-like activity.

The vasoconstrictor endothelin-1 (ET-1) is shown to have significant physiological effects on a unicellular organism, Tetrahymena pyriformis. These responses include: (1) A significant increase in intracellular [Ca2+] induced by 10(-10) M ET-1; (2) Increased chemotaxis, maximal at 10(-10) M; and (3) A small inhibition of proliferation at the 10(-13)-10(-12) M concentration range. Immunocytochemical detection of endogenous ET-1 using rabbit antibodies directed against human or porcine ET-1 indicates that this is a further example of the widening group of vertebrate hormones now known to be synthesized by Tetrahymena. These observations suggest that hormones are of considerable antiquity in their phylogenetic appearance and have been highly conserved throughout evolution.

Design, synthesis, and in vitro activity of novel drug delivery systems containing tuftsin derivatives and methotrexate.

During the past decade, biodegradable polymers or oligopeptides recognized by cell-surface receptors have been shown to increase drug specificity, lowering systemic drug toxicity in contrast to small-size fast-acting drugs. The goal of the present study was to develop anticancer bioconjugates based on chemotactic drug targeting (CDT). These constructs are composed of methotrexate (Mtx) attached to a tuftsin-like peptide carrier through an enzyme-labile pentapeptide spacer (GFLGC) and several copies of a chemotactic targeting moiety (H-TKPR, For-TKPR, H-TKPKG, and Ac-TKPKG). Carriers with targeting moieties in the branches were prepared by solid-phase synthesis using mixed Boc and Fmoc strategies. The drug molecule connected to an enzyme-labile spacer was attached to the branched oligopeptide in solution. In vitro chemotaxis, cellular uptake, and cytotoxicity assays were carried out on the MonoMac6 cell line. The most effective conjugates with H-TKPR or Ac-TKPKG targeting moieties in the branches, which have the most advantageous chemotactic properties, can be internalized rapidly, and these conjugates trigger higher toxic effect than the free drug (Mtx). The results suggest that our tuftsin-based drug delivery systems might be potential candidates for targeting cancer chemotherapy.

Method for determination of chemoattraction in Tetrahymena pyriformis

Earlier works presented a variety of techniques for detection of chemoattractants in ciliates. The present agar layer model and the used double-P cutter give an easy, reproducible way of quantitative evaluation of positive chemotaxis. Beside the two-channel chamber, the multichannel one seems to be also useful in characterization of different taxons or different chemoattractants.

EFFECTS OF DIPEPTIDES CONTAINING THE AMINO ACID, PROLINE ON THE CHEMOTAXIS OF TETRAHYMENA PYRIFORMIS. EVOLUTIONARY CONCLUSIONS ON THE FORMATION OF HORMONE RECEPTORS AND HORMONES

Our investigations demonstrate that proline‐containing dipeptides can provoke a chemosensory response from the unicellular Tetrahymena pyriformis The chemotactic effects of the dipeptides have a close relationship with the side chain and the lipophilicity of the amino‐terminal amino acid. Comparison of ‘mirror’ variants of proline‐containing dipeptides points to the fact that dipeptides with small side chain and non‐polar character amino acids (Gly‐Pro, Ala‐Pro) are preferred on the amino‐terminal end. In the case of amino acids with very variable side chains, small (Pro‐Gly) and the large side chain and non‐polar character amino acids (Pro‐Leu, Pro‐Phe) on the carboxyl‐terminal end can induce significant chemotactic responses. With valine on any terminus the proline‐containing dipeptide induced a weak repellent effect.

DIRECT CHEMOTACTIC EFFECT OF BRADYKININ AND RELATED PEPTIDES—SIGNIFICANCE OF AMINO- AND CARBOXYTERMINAL CHARACTER OF OLIGOPEPTIDES IN CHEMOTAXIS OF TETRAHYMENA PYRIFORMIS

The chemotactic character of the nonapeptide bradykinin (BK1–9) and its derivatives was studied in the eukaryotic ciliated model Tetrahymena pyriformis. The results demonstrate that BK1–9 has a direct and ligand‐specific chemoattractant effect (maximal at 10−11m) without any intermediate substance as is essential in some mammalian test systems. Evaluation of the chemotactic effect elicited by derivatives showed that the presence of N‐ and C‐terminal arginines can influence chemotactic potency of the molecule via expression of pyrrolidine and aromatic ring structures of terminal amino acid residues. Removal of the N‐terminal Arg (expression of Pro) results in a significant decrease in chemotaxis (BK2–9), while further truncation of the C‐terminal, causing expression of the aromatic ring of Phe (BK2–8), results in a highly chemoattractant variant. A single pyrrolidine ring on the C‐terminus BK1–7 also has a positive effect on the chemotactic character, however further truncation (BK1–6, BK1–5) causes the chemoattractant character to become chemorepellent. Study of chemotactic selection with BK derivatives supports our previous findings that only phylogenetically selected ligands or their close derivatives are able to induce long‐term selection with chemotaxis.

Characterization of chemotactic ability of peptides containing N‐formyl‐methionyl residues in Tetrahymena fMLP as a targeting ligand

The chemotactic effects of six formylated, putatively bacterial peptides (fMLP, fMLPP, fMMM, fMP, fMV, and fMS) were studied. From the set of six peptides, only fMLP (one of the most effective chemoattractant peptides in mammals) elicited a significant positive chemotactic response in the eukaryotic ciliate Tetrahymena pyriformis, while the other formylated ligands, e.g. fMMM (which is also effective in mammals), had neutral or antagonistic effects in Tetrahymena. A study of their amino acid sequences points to an, as yet obscure, interaction between C‐terminal f‐Met and N‐terminal aromatic Phe. Some optimal physicochemical characteristics (e.g. solvent exposed area, solubility) of the molecule may be responsible for this special feature of f‐MLP at such a low level of phylogeny. This means that the unicellular Tetrahymena is able to select between related molecules, giving high priority to the molecule that is the most chemoattractive in mammals. The results call attention to the possible presence of f‐Met receptors at a unicellular level and to the evolutionary conservation of chemotaxis‐activating processes.

Presence, uptake and localization of an immunoreactively interleukin 6 (IL-6)-like molecule in Tetrahymena pyriformis

The unicellular Tetrahymena and its medium contain immunoreactively interleukin 6 (IL‐6)‐like molecules (hereinafter IL‐6) in a measurable quantity in the 24 h‐old cultures. This protozoan takes up exogenously supplied IL‐6 very quickly, and this can be found in similar amounts in both the cells and the media after 1h. After 24h (48h cultures), an equal amount of IL‐6 is present in the control and IL‐6‐treated cells and their media. By 120h, cells which have not had their medium changed retained the same quantity of IL‐6 as the control; however less than half was found in IL‐6‐treated cells. In the medium of 120h‐old cultures, there was a reduction of IL‐6 content relative to the 24h content in the control; however, in the IL‐6‐treated cell culture medium, less than half of the level in the controls was found. Confocal microscopy demonstrated the localization of IL‐6 in/on the oral apparatus and basal bodies, and the nuclear envelope also showed moderate labelling. IL‐6 antibody binding was enhanced after IL‐6 pretreatment (hormonal imprinting). The experiments call attention to the presence of an IL‐6‐like molecule and its uptake at a very low level of phylogeny.

Chemotactic selection with insulin, di-iodotyrosine and histamine alters the phagocytotic responsiveness of Tetrahymena

Chemotactic selection is a method by which populations of cells exposed to ligands can be isolated and subsequently cultivated. We used Tetrahymena pyriformis GL cultures selected by chemotactic selection to insulin (10 nM), histamine (0.1 nM) and di-iodotyrosine (T2, 10 nM) to study the phagocytotic capacity under the induction of selector hormones. Our results show a long-lasting link between chemotactically selected cultures and phagocytotic activity. Cells selected to histamine produced the highest phagocytotic activity upon a second exposure to the selector hormone. T2 selection was also strongly effective, however, the phagocytosis stimulation was not specific to the hormone given later. Insulin selected sub-populations had different phagocytotic responses to the control substance itself, whereas histamine selected sub-populations seem to be heterogeneous in the phagocytotic response to histamine. For insulin, the increased endocytotic or metabolic activity was demonstrated by the lack of non-phagocytotic cells. These experiments call attention to the evolutionary role of selection in the later developing receptor-hormone relationship.