Cloning, localization, and axonemal function of Tetrahymena centrin.

Centrin, an EF hand Ca(2+) binding protein, has been cloned in Tetrahymena thermophila. It is a 167 amino acid protein of 19.4 kDa with a unique N-terminal region, coded by a single gene containing an 85-base pair intron. It has > 80% homology to other centrins and high homology to Tetrahymena EF hand proteins calmodulin, TCBP23, and TCBP25. Specific cellular localizations of the closely related Tetrahymena EF hand proteins are different from centrin. Centrin is localized to basal bodies, cortical fibers in oral apparatus and ciliary rootlets, the apical filament ring and to inner arm (14S) dynein (IAD) along the ciliary axoneme. The function of centrin in Ca(2+) control of IAD activity was explored using in vitro microtubule (MT) motility assays. Ca(2+) or the Ca(2+)-mimicking peptide CALP1, which binds EF hand proteins in the absence of Ca(2+), increased MT sliding velocity. Antibodies to centrin abrogated this increase. This is the first demonstration of a specific centrin function associated with axonemal dynein. It suggests that centrin is a key regulatory protein for Tetrahymena axonemal Ca(2+) responses, including ciliary reversal or chemotaxis.

The protozoan Tetrahymena as a bioindicator to screen bioactive substances.

Tetrahymena thermophila has been used as a "swimming receptor" to study its chemotaxis in the presence of various bioactive substances from herbal plants. Chemotaxis of this ciliated protozoan is, in part, controlled by a cyclic GMP-dependent protein kinase (PKG), which can adjust ciliary beating. In this paper, the effects of Ginkgo biloba extract (GBE) and its main functional constituents, terpene lactones, flavonol glycosides and aglycones, on the chemotaxis and PKG activity of ciliates, were systematically investigated. GBE and its constituents exerted significant inhibition of chemotaxis and PKG activity in cells of T. thermophila. The minimal concentrations to completely inhibit chemotaxis of T. thermophila were 12, 25, 50, 100, 300, 400, 400, 500 microM, 2 mg/ml for isorhamnetin, kaempferol, quercetin, myricetin, isoquercitrin, quercetin-3-beta-d-galactoside, rutin, quercitrin, and GBE, respectively. The IC(50) values for PKG were 14, 17, 20, 25, 186, 78 microM, 0.157 mg/ml for isorhamnetin, kaempferol, quercetin, myricetin, isoquercitrin, rutin and GBE, respectively. The results indicate that the chemotaxis inhibition by GBE and its constituents and their effects on PKG are similar. This suggests that T. thermophila may be a potential experimental organism for screening other bioactive substances.

Chemokinesis by tetrahymena in response to bacterial oligopeptides.

The ciliate Tetrahymena responds very efficiently by chemoattraction to a group of trichloroacetic acid-soluble oligopeptides isolated from a commercial bioprotein from Methanococcus. When fractionated by reversed phase C18-high-pressure liquid chromatography, this group of very efficient chemoattractants turned out to consist of a heterogeneous group of oligopeptides with molecular weight ranging from 0.2 to 1.5 kDa. The peptides were very rich in the following amino acids: aspartic acid, alanine, glutamic acid, proline, glycine, lysine, and arginine. The term chemokinesis is used throughout to emphasise that chemoattraction does not necessarily include an element of orientation of cells.

Ribosomal RNA transcription during macronuclear development ofTetrahymena thermophila.

Conjugation inTetrahymena, as in other ciliated protozoa is both a necessary tool for genetic studies and a potential model system in development. Conjugation is an ordered sequence of events which involves pair formation between two cells of different mating types followed by a precise sequence of nuclear events leading to the establishment of a new recombinant germinal nucleus (micronucleus) and then to the development of a new somatic nucleus (macronnucleus) from the germinal nucleus. The whole process takes about 20 h at 30°C and can be performed with large volumes of cells.The synthesis of ribosomal RNA during macronuclear development was studied in cultures of conjugatingTetrahymena thermophila by following the incorporation of3H-uridine into whole cells and purified ribosomal and pre-ribosomal RNA as well as by measuring bulk-RNA accumulation. In starving cultures and conjugating cultures refed with growth medium during late conjugation, some (background) ribosomal RNA synthesis was detectable 11-12 h after mixing the cells, which is the time when conjugating cells come apart but the macronnucleus is still developing. However, the major burst of rRNA accumulation occurred 13-18 h in refed conjugants.Observation of the conjugating cells by transmission electron microscopy showed that development of nucleoli took place in the macronuclear analagen concomitantly with the major burst of ribosomal RNA synthesis (13-18 h). A nucleolar organization similar to that found in vegetative cells was attained in the macronuclear anlagen 18 h after mixing of the cells.

Protein phosphatase 2A (PP2A) regulates interleukin-4-mediated STAT6 signaling.

Interleukin-4 (IL-4) plays a pivotal role in the induction and maintenance of allergy by promoting Th2 differentiation and B cell isotype switching to IgE. Studies on STAT6-deficient mice have demonstrated the essential role of STAT6 in mediating the biological functions of IL-4. IL-4 induces tyrosine phosphorylation of STAT6, which in turn leads to transcription of IL-4-specific genes. In addition, serine phosphorylation of STAT6 has recently been reported. Here we study the functional role of STAT6 serine phosphorylation and the kinases and phosphatases involved. We show that inhibition of protein phosphatase 2A (PP2A) induces serine phosphorylation of STAT6 and severely inhibits DNA binding of STAT6. In contrast, IL-4-induced tyrosine phosphorylation of Janus kinase-1 and STAT6 is not affected, suggesting that PP2A acts downstream of Janus kinases in IL-4 signaling. In conclusion, we provide the first evidence that PP2A plays a crucial role in the regulation of STAT6 function.