Use of E-64 cysteine protease inhibitor for the recombinant protein production in Tetrahymena thermophila.

Tetrahymena thermophila is an alternative organism for recombinant protein production. However, the production efficiency in T. thermophila is quite low mainly due to the rich cysteine proteases. In this study, we studied whether supplementation of the E-64 inhibitor to T. thermophila cultures increases the recombinant protein production efficiency without any toxic side effects. Our study showed that supplementation of E-64 had no lethal effects on T. thermophila cells in flask culture at 30 °C and 38 °C. In vitro protease activity analysis using secretome as protease enzyme source from E-64-supplemented cell cultures showed a reduced protein substrate degradation using bovine serum albumin, rituximab, and milk lactoglobulin proteins. E-64 also prevented proteolysis of the recombinantly produced and secreted TtmCherry2-sfGFP fusion protein at some level. This reduced inhibitory effect of E-64 could be due to genetic compensation of the inhibited proteases. As a result, the 5 µM concentration of E-64 was found to be a non-toxic protease inhibitory supplement to improve extracellular recombinant protein production efficiency in T. thermophila. This study suggests that the use of E-64 may increase the efficiency of extracellular recombinant protein production by continuously reducing extracellular cysteine protease activity during cultivation.

Inhibition of Mitochondrial ATP Synthesis and Regulation of Oxidative Stress Based on {SbW8 O30 } Determined by Single-Cell Proteomics Analysis.

The 10-nuclear heteroatom cluster modified {SbW8 O30 } was successfully synthesized and exhibited inhibitory activity (IC50 =0.29 µM). Based on proteomics analysis, Na4 Ni2 Sb2 W2 -SbW8 inhibited ATP production by affecting the expression of 16 related proteins, hindering metabolic functions in vivo and cell proliferation due to reactive oxygen species (ROS) stress. In particular, the low expression of FAD/FMN-binding redox enzymes (relative expression ratio of the experimental group to the control=0.43843) could be attributed to the redox mechanism of Na4 Ni2 Sb2 W2 -SbW8 , which was consistent with the effect of polyoxometalates (POMs) and FMN-binding proteins on ATP formation. An electrochemical study showed that Na4 Ni2 Sb2 W2 -SbW8 combined with FMN to form Na4 Ni2 Sb2 W2 -SbW8 -2FMN complex through a one-electron process of the W atoms. Na4 Ni2 Sb2 W2 -SbW8 acted as catalase and glutathione peroxidase to protect the cell from ROS stress, and the inhibition rates were 63.3 % at 1.77 µM of NADPH and 86.06 % at 10.62 µM of 2-hydroxyterephthalic acid. Overall, our results showed that POMs can be specific oxidative/antioxidant regulatory agents.

Occurrence of the fungus mycotoxin, ustiloxin A, in surface waters of paddy fields in Enshi, Hubei, China, and toxicity in Tetrahymena thermophila.

There has been an increasing incidence rate of rice false smut in global rice cultivation areas. However, there is a dearth of studies on the environmental concentrations and hazards of ustiloxin A (UA), which is the major mycotoxin produced by a pathogenic fungus of the rice false smut. Here, the concentrations of UA in the surface waters of two paddy fields located in Enshi city, Hubei province, China, were measured, and its toxicity in T. Thermophila was evaluated. This is the first study to detect UA in the surface waters of the two paddy fields, and the measured mean concentrations were 2.82 and 0.26 µg/L, respectively. Exposure to 2.19, 19.01 or 187.13 µg/L UA for 5 days significantly reduced the theoretical population and cell size of T. thermophila. Furthermore, treatment with 187.13 µg/L UA changed the percentages of T. thermophila cells in different cell-cycle stages, and with an increased malformation rate compared with the control, suggesting the disruption of the cell cycle. The expressions of 30 genes involved in the enriched proteasome pathway, 7 cyclin genes (cyc9, cyc10, cyc16, cyc22, cyc23, cyc26, cyc33) and 2 histone genes (mlh1 and hho1) were significantly down-regulated, which might be the modes of action responsible for the disruption of cell cycling due to UA exposure.

Extreme metal adapted, knockout and knockdown strains reveal a coordinated gene expression among different Tetrahymena thermophila metallothionein isoforms.

Metallothioneins (MT) constitute a superfamily of small cytosolic proteins that are able to bind metal cations through numerous cysteine (Cys) residues. Like other organisms the ciliate Tetrahymena thermophila presents several MT isoforms, which have been classified into two subfamilies (Cd- and Cu-metallothioneins). The main aim of this study was to examine the specific functions and transcriptional regulation of the five MT isoforms present in T. thermophila, by using several strains of this ciliate. After a laboratory evolution experiment over more than two years, three different T. thermophila strains adapted to extreme metal stress (Cd2+, Cu2+ or Pb2+) were obtained. In addition, three knockout and/or knockdown strains for different metallothionein (MT) genes were generated. These strains were then analyzed for expression of the individual MT isoforms. Our results provide a strong basis for assigning differential roles to the set of MT isoforms. MTT1 appears to have a key role in adaptation to Cd. In contrast, MTT2/4 are crucial for Cu-adaptation and MTT5 appears to be important for Pb-adaptation and might be considered as an "alarm" MT gene for responding to metal stress. Moreover, results indicate that likely a coordinated transcriptional regulation exists between the MT genes, particularly among MTT1, MTT5 and MTT2/4. MTT5 appears to be an essential gene, a first such report in any organism of an essential MT gene.

Ionophoric polyphenols selectively bind Cu(2+), display potent antioxidant and anti-amyloidogenic properties, and are non-toxic toward Tetrahymena thermophila.

Alzheimer's disease (AD) is the most common form of dementia affecting more than 28million people in the world. Only symptomatic treatments are currently available. Anticipated tri-fold increase of AD incidence in the next 50years has established the need to explore new possible treatments. Accumulation of extracellular amyloid-ß (Aß) plaques, intracellular tangles in the brain, and formation of reactive oxygen species (ROS) are the major hallmarks of the disease. The active role of some metal ions, especially Cu(2+), in promoting both Aß aggregation and reactive oxygen species formation has rendered ionophoric drugs as a promising treatment strategy. In this work, a series of 5 disease-modifying and multi-target ionophoric polyphenols (1-5), inspired on the structure of natural resveratrol, have been synthesized and characterized. All compounds bind Cu(2+) selectively over other biologically relevant metal ions. They form 2:1 (compound/Cu(2+)) complexes with association constants logKa 12-14 depending on the molecular design. Our results indicate that compounds 1-5 possess excellent antioxidant properties: they inhibit the Cu(2+)-catalyzed reactive oxygen species production between 47% and 100%, and they scavenge DPPH (1,1-diphenyl-2-picryl-hydrazyl) and AAPH (2,2'-azobis(2-amindino-propane)dihydrochloride) free radicals in general better than clioquinol, resveratrol and ascorbic acid. In addition, compounds 1-5 interact with Aß peptides and inhibit both the Cu(2+)-catalyzed aggregation and the self-assembly of Aß(1-40) up to a ∼92% extent. Interestingly, 1-5 are also able to disaggregate up to ∼91% of pre-formed Aß(1-40) aggregates. Furthermore, cytotoxic studies show remarkably low toxicity of 1-5 toward Tetrahymena thermophila with LD50 values higher than 150µM, comparable to non-toxic natural resveratrol.

Off-target effects of the septin drug forchlorfenuron on nonplant eukaryotes.

The septins are a family of GTP-binding proteins that form cytoskeletal filaments. Septins are highly conserved and evolutionarily ancient but are absent from land plants. The synthetic plant cytokinin forchlorfenuron (FCF) was shown previously to inhibit budding yeast cell division and induce ectopic septin structures (M. Iwase, S. Okada, T. Oguchi, and A. Toh-e, Genes Genet. Syst. 79:199-206, 2004, http://dx.doi.org/10.1266/ggs.79.199). Subsequent studies in a wide range of eukaryotes have concluded that FCF exclusively inhibits septin function, yet the mechanism of FCF action in nonplant cells remains poorly understood. Here, we report that the cellular effects of FCF are far more complex than previously described. The reported growth arrest of budding yeast cells treated with 1 mM FCF partly reflects sensitization caused by a bud4 mutation present in the W303 strain background. In wild-type (BUD4(+)) budding yeast, growth was inhibited at FCF concentrations that had no detectable effect on septin structure or function. Moreover, FCF severely inhibited the proliferation of fission yeast cells, in which septin function is nonessential. FCF induced fragmentation of budding yeast mitochondrial reticula and the loss of mitochondrial membrane potential. Mitochondria also fragmented in cultured mammalian cells treated with concentrations of FCF that previously were assumed to target septins only. Finally, FCF potently inhibited ciliation and motility and induced mitochondrial disorganization in Tetrahymena thermophila without apparent alterations in septin structure. None of these effects was consistent with the inhibition of septin function. Our findings point to nonseptin targets as major concerns when using FCF.

Carbon nanotube compared with carbon black: effects on bacterial survival against grazing by ciliates and antimicrobial treatments.

The ingestion and digestion of Escherichia coli by the ciliated protozoan, Tetrahymena thermophila, was investigated after an initial exposure to either water-soluble single-walled carbon nanotubes (SWNT) or to carbon black (CB). Both SWNT and CB were internalised and visible in food vacuoles of ciliates. When presented with E. coli expressing green-fluorescent protein (GFP), these ciliates internalised bacteria as well. However, ciliates that had first internalised SWNT but not CB subsequently externalised or egested vesicle-like structures with fluorescent bacteria inside. These egested bacteria were viable and less susceptible than planktonic E. coli to killing either by the antibiotic, chloramphenicol or the disinfectant, glutaraldehyde. These results suggest that SWNT can alter the intracellular trafficking of vesicles within ciliates, leading to bacterial prey being packaged externally and protected for a time from environmental killing, which could have implications for sewage treatment and for public health.

Effects of a new photoactivatable cationic porphyrin on ciliated protozoa and branchiopod crustaceans, potential components of freshwater ecosystems polluted by pathogenic agents and their vectors.

The increasing use of photosensitized processes for disinfection of microbiologically polluted waters requires a precise definition of the factors controlling the degree of photosensitivity in target and non-target organisms. In this regard, tests with protozoa and invertebrates which have a natural habitat in such waters may be used as first screening methods for the assessment of possible hazards for the ecosystem. A new cationic porphyrin, namely meso-tri(N-methyl-pyridyl)mono(N-dodecyl-pyridyl)porphine (C12), is tested in this work on the protozoan Ciliophora Colpoda inflata and Tetrahymena thermophila and the Crustacea Branchiopoda Artemia franciscana and Daphnia magna. The protocol involved 1 h incubation with porphyrin doses in the 0.1-10.0 µM range and subsequent irradiation with visible light at a fluence rate of 10 mW cm(-2). The results indicate that C12 porphyrin has a significant affinity for C. inflata and T. thermophila; this is also shown by fluorescence microscopic analyses. C. inflata cysts were resistant to the phototreatment up to a porphyrin dose of 0.6 µM. The effects of C12 on cysts have been evaluated at 3 and 24 h after the end of the phototreatment; a delay in the excystment process was observed. T. thermophila was fairly resistant to the phototreatment with C12 porphyrin. The data obtained with the two crustaceans indicated that the effects of dark- and photo-treatment with C12 need to be closely examined for every organism. A. franciscana is more resistant, probably owing to its ability to adapt to extreme conditions, while the high level of photosensitivity displayed by Daphnia magna represents a potential drawback, as this organism is often selected as a reference standard for assessing the environmental safety. Thus, while C12 photosensitisation can represent a useful tool for inducing a microbicidal or larvicidal action on polluted waters, the irradiation protocols must be carefully tailored to the nature of the specific water basin, and in particular to its biotic characteristics.

Preparation and biological effect of nucleotide-capped CdSe/ZnS quantum dots on Tetrahymena thermophila.

In this paper, we described the preparation and characterization of different types of modified CdSe/ZnS quantum dots (QDs) and explored the biological effects of QDs with different surface modifications on the whole growth of unicellular protozoan Tetrahymena thermophila BF(5) using a thermal activity monitor air isothermal microcalorimeter. Our results demonstrated that adenosine 5'-monophosphate (AMP) showed stronger interaction with QDs than other types of nucleotide. AMP-QDs could stimulate the growth of T. thermophila while mercaptoacetic acid-capped CdSe/ZnS quantum dots inhibited it. In addition, the population density determination and fluorescence imaging of T. thermophila BF(5) also confirmed the results obtained from microcalorimetry. It is believed that this approach will provide a more convenient methodology for the kinetics and thermodynamics of microorganism when coexisting with QDs in real time, and all of which are very significant to understanding the effect of QDs to organism.

Highly divergent mitochondrial ATP synthase complexes in Tetrahymena thermophila.

The F-type ATP synthase complex is a rotary nano-motor driven by proton motive force to synthesize ATP. Its F(1) sector catalyzes ATP synthesis, whereas the F(o) sector conducts the protons and provides a stator for the rotary action of the complex. Components of both F(1) and F(o) sectors are highly conserved across prokaryotes and eukaryotes. Therefore, it was a surprise that genes encoding the a and b subunits as well as other components of the F(o) sector were undetectable in the sequenced genomes of a variety of apicomplexan parasites. While the parasitic existence of these organisms could explain the apparent incomplete nature of ATP synthase in Apicomplexa, genes for these essential components were absent even in Tetrahymena thermophila, a free-living ciliate belonging to a sister clade of Apicomplexa, which demonstrates robust oxidative phosphorylation. This observation raises the possibility that the entire clade of Alveolata may have invented novel means to operate ATP synthase complexes. To assess this remarkable possibility, we have carried out an investigation of the ATP synthase from T. thermophila. Blue native polyacrylamide gel electrophoresis (BN-PAGE) revealed the ATP synthase to be present as a large complex. Structural study based on single particle electron microscopy analysis suggested the complex to be a dimer with several unique structures including an unusually large domain on the intermembrane side of the ATP synthase and novel domains flanking the c subunit rings. The two monomers were in a parallel configuration rather than the angled configuration previously observed in other organisms. Proteomic analyses of well-resolved ATP synthase complexes from 2-D BN/BN-PAGE identified orthologs of seven canonical ATP synthase subunits, and at least 13 novel proteins that constitute subunits apparently limited to the ciliate lineage. A mitochondrially encoded protein, Ymf66, with predicted eight transmembrane domains could be a substitute for the subunit a of the F(o) sector. The absence of genes encoding orthologs of the novel subunits even in apicomplexans suggests that the Tetrahymena ATP synthase, despite core similarities, is a unique enzyme exhibiting dramatic differences compared to the conventional complexes found in metazoan, fungal, and plant mitochondria, as well as in prokaryotes. These findings have significant implications for the origins and evolution of a central player in bioenergetics.

A pseudo-phytochelatin synthase in the ciliated protozoan Tetrahymena thermophila.

Phytochelatins (PCs) and metallothioneins (MTs) are the two major heavy metal chelating peptides in eukaryotes. We report here on the identification of a biosynthetically inactive pseudo-phytochelatin synthase enzyme (TtpsiPCS) in the ciliate Tetrahymena thermophila, the first of this kind (pseudo-PCS) to be described in eukaryotes. TtpsiPCS which resembles a true PCS at the N-terminal region, while it is most divergent in its Cys-poor C-terminal region, was found to be up-regulated under cadmium stress conditions. However, only glutathione (GSH) hydrolysis products, but not PCs, could be detected in extracts from Cd-treated cells. The latter feature is reminiscent of pseudo-PCS enzymes recently identified in cyanobacteria, which are also biosynthetically inactive, but capable to hydrolyze GSH.

Bioassays for evaluating the water-extractable genotoxic and toxic potential of soils polluted by metal smelters.

Physicochemical analyses of polluted soils are limited in their ability to determine all hazardous compounds, their bioavailability, and their combined effects on living organisms. Bioassays, on the other hand, can evaluate environmental quality more accurately. This study assesses the genotoxic potential of water extracts from soil polluted with metals (Pb, Cd, and Zn) by the former lead smelter in zerjav, Slovenia using comet assay with Tetrahymena thermophila and human hepatoma cells (HepG2). In addition, the toxicity of soil samples and their extracts was evaluated using Vibrio fischeri and delayed fluorescence of Lemna minor. Chemical analyses of metals using atomic absorption spectrophotometry (AAS) was performed for comparison. Measurements of the total metal concentrations showed that four of five plots near the former lead smelter were highly contaminated with Pb, Cd, and Zn, but the amount of metals in water/soil extracts was low at all the sampling plots. Genotoxicity was demonstrated using T. thermophila for the majority of the extracts, and HepG2 cells for only some of the extracts. Whereas V. fischeri indicated a gradual decrease in soil toxicity with greater distance from the smelter, the toxicity of extracts did not correlate with proximity. Low concentrations of metals in water extracts stimulated L. minor growth. The results indicate that comet assay with T. thermophila and HepG2 cells and the BSPT with V. fischeri are suitable protocols for screening the genotoxic and toxic potential of water/soil extracts by comet assay, whereas chemical analyses of total metal concentrations in soil do not solely suffice for evaluating metal pollution in the environment. Biological assays are thus crucial for risk assessment.

Synthesis and an evaluation of the bioactivity of the C-glycoside of pseudopterosin A methyl ether.

The Suzuki-Miyaura cross-coupling protocol was applied to the synthesis of 1a, the C-glycoside analogue of PsA methyl ether. This marks the first construction of a C-glycoside for this class of marine natural products, thereby offering an opportunity to compare its bioactivity to the natural substances. Its activity profile resembled that of PsA (1) and PsA O-methyl ether (1b) when assayed for its anti-inflammatory activity and its ability to inhibit phagocytosis. We conclude that the intact structure is present when a pseudopterosin expresses its anti-inflammatory and phagocytosis inhibitory properties and that they are, therefore, not likely to be prodrugs. Results show that 1a is an effective binding agent toward the A2A and A3 adenosine receptors, displaying IC50 values of 20 and 10 microM, respectively.

A comparison of the polycation receptors of Paramecium tetraurelia and Tetrahymena thermophila.

Chemorepellents are compounds that cause ciliated protozoans to reorient their swimming direction. A number of chemorepellents have been studied in the ciliated protozoans, Paramecium and Tetrahymena. Chemorepellents, such as polycations, cause the organism to exhibit "avoidance behavior," a swimming behavior characterized by jerky movements and other deviations from normal forward swimming, which result from ciliary reversal. One well-characterized chemorepellent pathway in Tetrahymena is that of the proposed polycation receptor that is activated by lysozyme and pituitary adenylate cyclase activating polypeptide (PACAP). In this study, we compare the response of Paramecium to the chemorepellents lysozyme, vasoactive intestinal peptide (VIP), and PACAP to the previously studied polycation response in Tetrahymena. Our results indicate that lysozyme, VIP, and PACAP are all chemorepellents in Paramecium, just as they are in Tetrahymena. However, the signaling pathways involved appear to be different. While previous pharmacological characterization indicates that G-proteins are involved in polycation signaling in Tetrahymena, we present evidence that similar reception in Paramecium involves activation of a tyrosine kinase pathway in order for lysozyme avoidance to occur. Polycation responses of both organisms are inhibited by neomycin sulfate. While PACAP is the most effective of the three chemorepellents in Tetrahymena, lysozyme is the most effective chemorepellent in Paramecium.

Flow cytometry assessment of cytotoxicity and reactive oxygen species generation by single and binary mixtures of cadmium, zinc and copper on populations of the ciliated protozoan Tetrahymena thermophila.

In the present study, we have assessed by flow cytometry, the cytotoxicity of the heavy metals Cd, Zn and Cu on populations of the ciliated protozoan Tetrahymena thermophila. The obtained LC(50) for these metals was estimated as 0.195mgCdl(-1), 3.58mgZnl(-1) and 0.47mgCul(-1), respectively. As a result, the toxicity rank for this eukaryotic microorganism is Cd>Cu>>Zn. Using the same methodology and the Concentration Addition approach, the toxicity of binary mixtures of these metals was evaluated in order to detect the type of interaction between these metals. Results indicated that antagonism is the predominant interaction but it can change to additivity or even to synergism at high metal concentrations. Besides, the concentration ratio between metals plays also a crucial role in determining the type of metallic interaction, at least in Tetrahymena. Cytotoxicity data from single and bimetallic mixtures have been compared with those from selected microalgae, other species of ciliates, fish and mammalian cell lines and metazoan. By other hand, we have detected the mitochondrial generation of peroxides induced by both single and binary treatments with Cd, Zn and Cu on populations of Tetrahymena, using the specific fluorophore dyhidrorhodamine. The nature and concentrations of metal as well as the metallic ratio were important factors in reactive oxygen species production. All results found in T. thermophila are compared with previous reports in other organisms and, some explanations and hypothesis to support results are given, including the involvement of metallothioneins as antioxidants and their role in the binding of metal cations.

Pseudopterosin A inhibits phagocytosis and alters intracellular calcium turnover in a pertussis toxin sensitive site in Tetrahymena thermophila.

The free living ciliate Tetrahymena thermophila was chosen as a cellular model in order to investigate the mode of action of the anti-inflammatory marine natural product Pseudopterosin A (PsA). In this paper we present evidence that PsA inhibits phagosome formation (KD=10.5 microM) and triggers a discrete intracellular calcium release (depletion) from a site in T. thermophila cells (KD=6.4 microM). Pre-treatment with the Gi/o protein inhibitor, pertussis toxin (PTX), inhibits PsA activity of both responses providing pharmacological evidence that the site of action for PsA is at a PTX sensitive G protein or a G protein coupled receptor (GPCR). Addition of extracellular calcium induced a concentration dependent increase in the incidence of phagosome formation (KD=30.3 microM) and was blocked by PsA pre-treatment. This particular effect of PsA on extracellular calcium was not blocked by PTX pre-treatment.

Dephosphorylation of inner arm 1 is associated with ciliary reversals in Tetrahymena thermophila.

In many organisms, depolarizing stimuli cause an increase in intraciliary Ca2+, which results in reversal of ciliary beat direction and backward swimming. The mechanism by which an increase in intraciliary Ca2+ causes ciliary reversal is not known. Here we show that Tetrahymena cells treated with okadaic acid or cantharidin to inhibit protein phosphatases do not swim backwards in response to depolarizing stimuli. We also show that both okadaic acid and cantharidin inhibit backward swimming in reactivated, extracted cell models treated with Ca2+. In contrast, treatment of whole cells or extracted cell models with protein kinase inhibitors has no effect on backward swimming. These results suggest that a component of the axonemal machinery is dephosphorylated during ciliary reversal. The phosphorylation state of inner arm dynein 1 (I1) was determined before and after cells were exposed to depolarizing conditions that induce ciliary reversal. An I1 intermediate chain is phosphorylated in forward swimming cells but is dephosphorylated in cells treated with a depolarizing stimulus. Our results suggest that dephosphorylation of Tetrahymena inner arm dynein 1 may be an essential part of the mechanism of ciliary reversal in response to increased intraciliary Ca2+.

Study of the environmental hazard caused by the oil shale industry solid waste.

The environmental hazard was studied of eight soil and solid waste samples originating from a region of Estonia heavily polluted by the oil shale industry. The samples were contaminated mainly with oil products (up to 7231mg/kg) and polycyclic aromatic hydrocarbons (PAHs; up to 434mg/kg). Concentrations of heavy metals and water-extractable phenols were low. The toxicities of the aqueous extracts of solid-phase samples were evaluated by using a battery of Toxkit tests (involving crustaceans, protozoa, rotifers and algae). Waste rock and fresh semi-coke were classified as of "high acute toxic hazard", whereas aged semi-coke and most of the polluted soils were classified as of "acute toxic hazard". Analysis of the soil slurries by using the photobacterial solid-phase flash assay showed the presence of particle-bound toxicity in most samples. In the case of four samples out of the eight, chemical and toxicological evaluations both showed that the levels of PAHs, oil products or both exceeded their respective permitted limit values for the living zone (20mg PAHs/kg and 500mg oil products/kg); the toxicity tests showed a toxic hazard. However, in the case of three samples, the chemical and toxicological hazard predictions differed markedly: polluted soil from the Erra River bank contained 2334mg oil/kg, but did not show any water-extractable toxicity. In contrast, spent rock and aged semi-coke that contained none of the pollutants in hazardous concentrations, showed adverse effects in toxicity tests. The environmental hazard of solid waste deposits from the oil shale industry needs further assessment.

A regulatory light chain of ciliary outer arm dynein in Tetrahymena thermophila.

Ciliary beat frequency is primarily regulated by outer arm dyneins (22 S dynein). Chilcote and Johnson (Chilcote, T. J., and Johnson, K. A. (1990) J. Biol. Chem. 256, 17257-17266) previously studied isolated Tetrahymena 22 S dynein, identifying a protein p34, which showed cAMP-dependent phosphorylation. Here, we characterize the molecular biochemistry of p34 further, demonstrating that it is the functional ortholog of the 22 S dynein regulatory light chain, p29, in Paramecium. p34, thiophosphorylated in isolated axonemes in the presence of cAMP, co-purified with 22 S dynein and not with inner arm dynein (14 S dynein). Isolated 22 S dynein containing phosphorylated p34 showed approximately 70% increase in in vitro microtubule translocation velocity compared with its unphosphorylated counterpart. Extracted p34 rebound to isolated 22 S dynein from either Tetrahymena or Paramecium but not to 14 S dynein from either ciliate. Binding of radiolabeled p34 to 22 S dynein was competitive with p29. Phosphorylated p34 was not present in axonemes isolated from a mutant lacking outer arms. Two-dimensional gel electrophoresis followed by phosphorimaging revealed at least five phosphorylated p34-related spots, consistent with multiple phosphorylation sites in p34 or perhaps multiple isoforms of p34. These new features suggest that a class of outer arm dynein light chains including p34 regulates microtubule sliding velocity and consequently ciliary beat frequency through phosphorylation.

PACAP-38 is a chemorepellent and an agonist for the lysozyme receptor in tetrahymena thermophila.

Pituitary adenylate cyclase activating peptide (PACAP-38) is a peptide hormone which functions in many mammalian systems, including the nervous and digestive systems. Using in vivo behavioral studies, we have found that this hormone functions as a chemore-pellent in Tetrahymena thermophila with an EC50 of 10 nM. Cells previously adapted to PACAP-38 were found to be adapted to lysozyme and vice versa. Furthermore, the in vivo behavioral activity of PACAP-38 was blocked by addition of the anti-lysozyme receptor antibody, 5545. Chemorepellent activity of PACAP-38 was also inhibited by the addition of neomycin sulfate (inhibition constant Ki = 0.080 micromol x l(-1)), a competitive inhibitor of lysozyme binding to its receptor. PACAP-38 is a more potent and specific agonist for the lysozyme receptor than either intact lysozyme or CB2, a 24-amino acid fragment of lysozyme.