Induction of oxidative stress, apoptosis and DNA damage by koumine in Tetrahymena thermophila.

Koumine is a component of the Chinese medicinal herb Gelsemium elegans and is toxic to vertebrates. We used the ciliate Tetrahymena thermophila as a model to evaluate the toxic effects of this indole alkaloid in eukaryotic microorganisms. Koumine inhibited T. thermophila growth and viability in a dose-dependent manner. Moreover, this drug produced oxidative stress in T. thermophila cells and expressions of antioxidant enzymes were significantly elevated at high koumine levels (p < 0.05). Koumine also caused significant levels of apoptosis (p < 0.05) and induced DNA damage in a dose-dependent manner. Mitophagic vacuoles were present in cells indicating induction of autophagy by this drug. Expression of ATG7, MTT2/4, CYP1 and HSP70 as well as the MAP kinase pathway gene MPK1 and MPK3 were significantly altered after exposed to koumine. This study represents a preliminary toxicological evaluation of koumine in the single celled eukaryote T. thermophila.

In vivo synthesis of nano-selenium by Tetrahymena thermophila SB210.

Nano-selenium has a great potential to be used in chemical, biological, medical and environmental fields. Biological methods for nano-selenium synthesis have attracted wide interests, because they can be operated at ambient temperature and pressure without complicated equipments. In this work, a protozoa, Tetrahymena thermophila (T. thermophila) SB210, was used to in vivo synthesize nano-selenium. The biosynthesized nano-selenium was characterized using transmission electron microscopy, energy dispersive X-ray spectroscopy and Raman spectroscopy. The synthesized amorphous spherical selenium nanoparticles had diameters of 50-500nm with the coexistence of irregular nano-selenium. The expressions of glutathione (GSH) synthesis related gene glutathione synthase, cysteine-rich protein metallothionein related gene metallothionein-1 and [2Fe-2S] cluster-binding protein related gene were up-regulated in the nano-selenium producing group. Also, the subsequent GSH detection and in vitro synthesis experimental results suggest the three proteins were likely to be involved in the nano-selenium synthesis process.

Checkpoint Activation of an Unconventional DNA Replication Program in Tetrahymena.

The intra-S phase checkpoint kinase of metazoa and yeast, ATR/MEC1, protects chromosomes from DNA damage and replication stress by phosphorylating subunits of the replicative helicase, MCM2-7. Here we describe an unprecedented ATR-dependent pathway in Tetrahymena thermophila in which the essential pre-replicative complex proteins, Orc1p, Orc2p and Mcm6p are degraded in hydroxyurea-treated S phase cells. Chromosomes undergo global changes during HU-arrest, including phosphorylation of histone H2A.X, deacetylation of histone H3, and an apparent diminution in DNA content that can be blocked by the deacetylase inhibitor sodium butyrate. Most remarkably, the cell cycle rapidly resumes upon hydroxyurea removal, and the entire genome is replicated prior to replenishment of ORC and MCMs. While stalled replication forks are elongated under these conditions, DNA fiber imaging revealed that most replicating molecules are produced by new initiation events. Furthermore, the sole origin in the ribosomal DNA minichromosome is inactive and replication appears to initiate near the rRNA promoter. The collective data raise the possibility that replication initiation occurs by an ORC-independent mechanism during the recovery from HU-induced replication stress.

Hints for metal-preference protein sequence determinants: different metal binding features of the five tetrahymena thermophila metallothioneins.

The metal binding preference of metallothioneins (MTs) groups them in two extreme subsets, the Zn/Cd- and the Cu-thioneins. Ciliates harbor the largest MT gene/protein family reported so far, including 5 paralogs that exhibit relatively low sequence similarity, excepting MTT2 and MTT4. In Tetrahymena thermophila, three MTs (MTT1, MTT3 and MTT5) were considered Cd-thioneins and two (MTT2 and MTT4) Cu-thioneins, according to gene expression inducibility and phylogenetic analysis. In this study, the metal-binding abilities of the five MTT proteins were characterized, to obtain information about the folding and stability of their cognate- and non-cognate metal complexes, and to characterize the T. thermophila MT system at protein level. Hence, the five MTTs were recombinantly synthesized as Zn(2+)-, Cd(2+)- or Cu(+)-complexes, which were analyzed by electrospray mass spectrometry (ESI-MS), circular dichroism (CD), and UV-vis spectrophotometry. Among the Cd-thioneins, MTT1 and MTT5 were optimal for Cd(2+) coordination, yielding unique Cd17- and Cd8- complexes, respectively. When binding Zn(2+), they rendered a mixture of Zn-species. Only MTT5 was capable to coordinate Cu(+), although yielding heteronuclear Zn-, Cu-species or highly unstable Cu-homometallic species. MTT3 exhibited poor binding abilities both for Cd(2+) and for Cu(+), and although not optimally, it yielded the best result when coordinating Zn(2+). The two Cu-thioneins, MTT2 and MTT4 isoforms formed homometallic Cu-complexes (major Cu20-MTT) upon synthesis in Cu-supplemented hosts. Contrarily, they were unable to fold into stable Cd-complexes, while Zn-MTT species were only recovered for MTT4 (major Zn10-MTT4). Thus, the metal binding preferences of the five T. thermophila MTs correlate well with their previous classification as Cd- and Cu-thioneins, and globally, they can be classified from Zn/Cd- to Cu-thioneins according to the gradation: MTT1>MTT5>MTT3>MTT4>MTT2. The main mechanisms underlying the evolution and specialization of the MTT metal binding preferences may have been internal tandem duplications, presence of doublet and triplet Cys patterns in Zn/Cd-thioneins, and optimization of site specific amino acid determinants (Lys for Zn/Cd- and Asn for Cu-coordination).

Bioenergetic investigation of the effects of La(III) and Ca(II) on the metabolic activity of Tetrahymena thermophila BF5.

The heat flux of Tetrahymena thermophila BF5 during growth and the effects of La(3+) and Ca(2+) on them were investigated with microcalorimetry; simultaneously, morphological changes of T. thermophila were obtained by light microscope. La(3+) in low concentration (0-5.0 x 10(-4) mol/l) remarkably stimulated T. thermophila metabolism, but high dose of La(3+) (5.8-8.6 x 10(-4) mol/l) restrained it in a linear manner with IC(50) being 7.2 x 10(-4) mol/l. In contrast, low concentration of Ca(2+) did not manifest obvious stimulation on T. thermophila metabolism; moreover, the IC(50) of Ca(2+) was much higher than that of La(3+). Low concentration of La(3+) did not lead to changes in appearance of T. thermophila, but low dose of Ca(2+) clearly promoted the cell proliferation. In addition, the morphological changes of T. thermophila evoked by high concentrations of La(3+) and Ca(2+) were consistent with relevant microcalorimetric results. It is concluded that La and Ca influence T. thermophila via different pathways, and La represents toxic action rather than Ca analogy.

Starvation-induced cleavage of the tRNA anticodon loop in Tetrahymena thermophila.

Amino acid deprivation triggers dramatic physiological responses in all organisms, altering both the synthesis and destruction of RNA and protein. Here we describe, using the ciliate Tetrahymena thermophila, a previously unidentified response to amino acid deprivation in which mature transfer RNA (tRNA) is cleaved in the anticodon loop. We observed that anticodon loop cleavage affects a small fraction of most or all tRNA sequences. Accumulation of cleaved tRNA is temporally coordinated with the morphological and metabolic changes of adaptation to starvation. The starvation-induced endonucleolytic cleavage activity targets tRNAs that have undergone maturation by 5' and 3' end processing and base modification. Curiously, the majority of cleaved tRNAs lack the 3' terminal CCA nucleotides required for aminoacylation. Starvation-induced tRNA cleavage is inhibited in the presence of essential amino acids, independent of the persistence of other starvation-induced responses. Our findings suggest that anticodon loop cleavage may reduce the accumulation of uncharged tRNAs as part of a specific response induced by amino acid starvation.