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V Leick

Publications and source records attributed to V Leick.

At least 19 recordsLinked to original sources
PubMed2001

Insulin/FGF-binding ciliary membrane glycoprotein from Tetrahymena.

Triton X-100 extracted ciliary membrane protein from isolated cilia, prepared from the protozoon Tetrahymena thermophila, were fractionated by affinity chromatography on columns with covalently bound fibroblast growth factor (FGF), insulin, or concanavalin A (ConA), respectively. The eluted proteins were further analyzed by electrophoresis on sodium dodecyl sulfate polyacrylamide gels, isoelectric focusing, and by immunoblotting techniques using antibodies against the FGF receptor, platetelet derived growth factor (PDGF) receptor alpha-subunit, and insulin receptor beta-subunit. The particular antibodies were chosen because the peptides PDGF, FGF, insulin, and ConA are chemoattractants in this organism and corresponding binding (receptor) proteins could be expected to be identified. A 66 kDa protein fraction was eluted from the FGF-MiniLeak agarose, insulin-MiniLeak agarose and ConA sepharose. This fraction responded in Western immunoblots to an antibody against the beta-subunit of the human insulin receptor, to an antibody against the PDGF receptor (PDGFR) and also to an antibody against the bovine FGF receptor (FGFR) that is known, in other systems, to inhibit FGF binding to its receptor. When analyzed by SDS-PAGE and stained with Coomassie blue the 66 kDa fraction appeared as a single component. However, in some experiments it appeared more heterogeneous when stained with silver indicating the presence of minor components that may be a procedural artifact or isoforms of the same glycoprotein. The 66 kDa protein(s) migrated in isoelectric focusing with a pI of 7.4. The results are discussed in terms of the possible role of the 66 kDa glycoprotein as a protein involved in peptide-mediated cell signalling.

Animals↗
PubMed1999

Inhibition of protein phosphatase 2A induces serine/threonine phosphorylation, subcellular redistribution, and functional inhibition of STAT3.

Signal transducers and activators of transcription (STATs) are rapidly phosphorylated on tyrosine residues in response to cytokine and growth factor stimulation of cell surface receptors. STATs hereafter are translocated to the nucleus where they act as transcription factors. Recent reports suggest that serine phosphorylation of STATs also is involved in the regulation of STAT-mediated gene transcription. Here, we studied the role of serine/threonine phosphatases in STAT3 signaling in human antigen-specific CD4(+) T cell lines and cutaneous T cell lymphoma lines, expressing a constitutively activated STAT3. We show that an inhibitor of protein phosphatases (PPs) PP1/PP2A, calyculin A, induces (i) phosphorylation of STAT3 on serine and threonine residues, (ii) inhibition of STAT3 tyrosine phosphorylation and DNA binding activity, and (iii) relocation of STAT3 from the nucleus to the cytoplasm. Similar results were obtained with other PP2A inhibitors (okadaic acid, endothall thioanhydride) but not with inhibitors of PP1 (tautomycin) or PP2B (cyclosporine A). Pretreatment with the broad serine/threonine kinase inhibitor staurosporine partly blocked the calyculin A-induced STAT3 phosphorylation, whereas inhibitors of serine/threonine kinases, such as mitogen-activated protein kinase-1 extracellular-regulated kinase-kinase, mitogen-activated protein p38 kinase, and phosphatidylinositol 3-kinase, did not. In conclusion, we provide evidence that PP2A plays a crucial role in the regulation of STAT3 phosphorylation and subcellular distribution in T cells. Moreover, our findings suggest that the level of STAT3 phosphorylation is balanced between a staurosporine-sensitive kinase(s) and PP2A.

CD4-Positive T-Lymphocytes↗
PubMed1998

Signaling in unicellular eukaryotes.

Aspects of intercellular and intracellular signaling systems in cell survival, proliferation, differentiation, chemosensory behavior, and programmed cell death in free-living unicellular eukaryotes have been reviewed. Comparisons have been made with both bacteria and metazoa. The central organisms were flagellates (Trypanosoma, Leishmania, and Crithidia), slime molds (Dictyostelium), yeast cells (Saccharomyces cerevisiae), and ciliates (Paramecium, Euplotes, and Tetrahymena). There are two novel aspects in this review. First, cellular responses are viewed in an evolutionary perspective, rather than from the more prevailing one, in which the unicellular eukaryotes are seen by the mammalian organisms. Second, results obtained with cell cultures in minimal, chemically defined nutrient media at low cell densities where intercellular signaling is strongly reduced are discussed. These results shed light on control mechanisms and their cooperation inside the living cell. Intracellular systems have many common features in unicellular and multicellular organisms.

Animals↗
PubMed1994

Physiological parameters affecting the chemosensory response of Tetrahymena.

We have investigated the significance of a number of physiological parameters in the preparation of cells for experiments on chemokinesis in Tetrahymena. The study comprises (1) growth state of the cells, (2) composition of the starvation medium, (3) concentration of cells during starvation, (4) oxygen saturation of the starvation medium, (5) temperature during starvation, and (6) starvation period. By controlling the physiological state of the cells, we significantly improved the reproducibility of the results obtained in assays for chemokinesis in Tetrahymena. In short, cells optimal for chemokinesis at an assay temperature of 28 degrees C should be starved from the exponential growth phase in a concentration below 2 x 10(5) cells ml-1 for 10-20 h. The surface-to-volume ratio of the starvation medium--water or Hepes buffer--should be about 5 cm-1 (or more) to ensure more than 95% oxygen saturation of the starvation medium. Maximal chemosensory responses were obtained if the cells were starved at 21 degrees C. The chemokinetic potential of the cells decreased significantly, as did the levels of the ratio of ATP to ADP, if cells were starved at higher temperatures. A tentative correlation between the ATP level in the cells and the chemosensory potential of the cells has been found. We suggest that chemokinesis is a constant quality of Tetrahymena, because we found no sign that prolonged starvation or other changes applied to the cells produced an up-regulation of the chemosensory response. Apparently, starvation is obligatory only to remove the growth medium (which is itself a very potent attractant), thereby making the cells sensitive to the chemoattractants.

Animals↗
PubMed1994

An improved quantitative assay for chemokinesis in Tetrahymena.

This paper presents a quantitative and sensitive assay for the measurement of chemosensory behavior in Tetrahymena. The two-phase assay is easy to perform in large quantities, so a variety of compounds can be screened under comparable conditions. A suspension of 2 x 10(5) cells ml-1 (the upper phase) is starved for 20-40 h and then gently placed on top of a 5% solution of Metrizamide (the lower phase) in a disposable microcuvette. The optical density of the lower phase is monitored at 600 nm with an automated spectrophotometer at selected time points. Optimum sensitivity of the assay is achieved when the cells slowly but continuously enter the lower phase, so that about 5% of them will be in the lower phase within 30 min. Optimal chemosensory responses occurred in Tetrahymena thermophila at about 25 degrees C. The response was delayed at 15 degrees C and markedly reduced at 35 degrees C. The data suggest three bases for quantifying the response in the assay: (1) initial slope of the absorbance versus time; (2) final maximal absorbance within the time period of measurement; and (3) signal-to-noise ratio (S/N) at a fixed time. We have quantified--in terms of S/N--the chemosensory responses in Tetrahymena for the following compounds: beta-endorphin, fibroblast growth factor, insulin, and platelet-derived growth factor (PDGF); these substances were active in nanomolar concentrations, and the maximal S/N was between 3 and 5.1. Acetylcholine was active only in millimolar concentrations; maximal S/N was 4.1 at 1 mM.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Chemosensory behaviour of Tetrahymena.

Free swimming cells of the ciliated protozoan Tetrahymena are attracted to certain chemicals by chemokinesis. However, a special type of chemotaxis in response to a chemical gradient is found in cells gliding very slowly in semisolid media. In contrast to classical chemotaxis by leukocytes, which is solely positive towards chemo-attractants, the oriented chemokinesis by gliding Tetrahymena involves both positive and negative elements. The major chemo-attractants are peptides and/or proteins, and they may be compounds which signal the presence of food in the natural environment of this freshwater phagotroph.

Animals↗
PubMed1992

Chemotactic properties, cellular binding and uptake of peptides and peptide derivatives: studies with Tetrahymena thermophila.

Receptor-mediated binding of leukocyte chemotactic peptide, N-formylMet-Leu-Phe (fMLP), occurs in the ciliated protozoon Tetrahymena thermophila. In vivo labelling of the cells with N-formylMet-Leu-[3H]Phe ([3H]fMLP) shows that the cells bind the ligand with high affinity (KD = 4 x 10(-9) M to 1 x 10(-8) M). Moreover, Scatchard transformations of the binding data show that there are about 5 x 10(5) binding sites per cell on the cell surface. Two fluorescent derivatives of leukocyte chemotactic peptide, N-dansylMet-Leu-Phe (dansMLP) and N-formylMet-Leu-Phe-(N-dansyl-)Lys (fMLPdanLys) compete for the N-formylMet-Leu-Phe (fMLP) binding sites on the cell surface. Moreover, both derivatives have retained significant chemoattracting potentials. Fluorescence from dansMLP, but not from fMLPdansLys and dansyl-beta-endorphin, is internalized preferentially into small vesicles. The differences may, however, reflect that the fluorescence from the dansyl group is strongly quenched by a hydrophilic microenvironment when using the two latter peptide derivatives. In contrast, the dansyl group from dansMLP must be assumed to be embedded in a hydrophobic microenvironment in the vesicular membrane or membrane protein. Rhodamine-labelled bovine serum albumin, egg albumin and cytochrome c as well as dansylated bovine serum albumin, which are poor chemoattractants, are preferentially seen to be internalized into large vesicles (food vacuoles).

Amino Acid Sequence↗
PubMed1990

A paper membrane filter assay for ciliate chemoattraction.

A quantitative bioassay for ciliate chemoattraction based on the Boyden assay is described with the ciliates Tetrahymena thermophila and Tetrahymena pyriformis as test organisms. A chamber is separated into two compartments by a Whatman 3MM filter, and a suspension of starved cells (approximately 10(5) cells/ml) is placed in one compartment and a solution containing attractant in the other. The gradient of chemoattractant across the filter causes the cells to swim through the filter into the attractant-containing compartment where their appearance is determined by electronic cell counting. The assay described is convenient with a signal-to-noise ratio of approximately 10. It is shown here to work with the attractants proteose peptone and platelet-derived growth factor.

Animals↗
PubMed1985

Photoreaction of 8-methoxypsoralen with yeast-tRNAPhe. Identification of the major reaction sites.

Yeast tRNAPhe was photoreacted with [3H]8-methoxypsoralen and the product was digested with ribonuclease T1, ribonuclease A or a combination of the two or cleaved with sodium borohydride/aniline. The oligonucleotides from these digestions were analyzed by polyacrylamide gel electrophoresis or high-pressure liquid chromatography and the psoralen-containing fragments were identified. The results indicate that one major and two minor photoreaction sites for 8-methoxypsoralen exist in yeast tRNAPhe. The major site (containing about 55% of the label) was determined as U50 in the T psi arm of the tRNA molecule while the minor sites were assigned to U59 (30% of the label) and C70 (15%) respectively. Our results suggest that psoralens may be used as photoprobes for studying conformational changes in tRNA molecules.

Aniline Compounds↗
PubMed1984

Platelet-derived growth factor stimulates chemotaxis and nucleic acid synthesis in the protozoan Tetrahymena.

Platelet-derived growth factor (PDGF) is in concentrations of a few nanograms per ml a very active chemoattractant for the free-living ciliated protozoan Tetrahymena; at the same time it induces a rapid increase in incorporation of radioactive nucleic acid precursors into RNA and DNA. We find it remarkable that this lower eukaryote responds to platelet-derived growth factor in very much the same way as fibroblastic cells.

Animals↗
PubMed1983

A quantitative assay for ciliate chemotaxis.

A quantitative bioassay for ciliate chemotaxis based on the capillary principle is described using Tetrahymena thermophila as test organism. The attractant-containing assay tube designed for the bioassay attracts up to 4 X 10(4) cells in 2 h which makes electronic cell counting of the chemotactic response feasible. The attractants used are solutions of proteose peptone and yeast extract which also are growth media for this organism.

Cell Count↗
PubMed1979

Analogues of [3H] chloramphenicol for photoaffinity labeling.

The synthesis of [3H]chloramphenicol and its erythro-diastereoisomer with specific activities of 1.25 Ci/mmol, and the further transformation of the [3H]chloramphenicol to a series of azido and diazo-substituted derivatives are described. The antibiotic activity of the compounds was considered insufficient for their use as photoaffinity labels.

Affinity Labels↗
PubMed1979

DNA interstrand crosslinks visualized by electron microscopy in PUVA-treated psoriasis.

An electron microscopic method has been used to visualize crosslinks after total denaturation of DNA isolated from epidermis and dermis in patients with psoriasis treated with 8-methoxypsoralen (8-MOP) and irradiation with ultraviolet light at 360 nm (PUVA treatment). This technique enabled accurate measurement of the number and density of DNA interstrand crosslinks. 30 biopsies were studied and a total of 9503 DNA molecules were scored in the electron microscope. 6 patients were treated topically with 8-MOP and 10 were on systemic treatment and biopsies were obtained immediately after irradiation. 1% of the total DNA molecules examined contained 3 or more crosslinks. The overall frequency of crosslinks was almost identical in the epidermis (1.1%) and in the dermis (0.9%) and, furthermore, virtually the same in patients on topical and systemic PUVA treatment. The total number of crosslinks was of the same magnitude as those previously found by us in normal human skin. Thus, though the formation of interstrand DNA crosslinks can be considered as an indicator of damage of the genetic material, we were unable to demonstrate this as being a consequence of PUVA treatment using 8-MOP.

Adult↗