SFTG international collaborative study on in vitro micronucleus test IV. Using CHL cells

In this report, are presented the results of an international collaborative study on the in vitro micronucleus assay, using CHL cells. Fourteen laboratories participated in this study which was coordinated by an organizing committee supported by the SFTG (the French branch of the European Environmental Mutagen Society). Nine coded substances, having different modes of action and at different levels were assessed in the in vitro micronucleus test, using a common protocol. Mitomycin C was used as a positive control. In order to help to define a standard protocol on CHL cells, short and long treatment periods followed by various recovery times, with or without cytochalasin B, were compared. After an evaluation of the acceptability of the assays, the tested chemicals were classified as negative, positive or equivocal. Mannitol and clofibrate were judged as negative in all treatment schedules. Bleomycin was positive in all the treatment schedules, with an increase in the number of micronucleated cells in both mononucleate and binucleate cells when using cytochalasin B. This was also shown for the aneugens colchicine, diethylstilboestrol and griseofulvin, as expected. Urethane was judged as equivocal only after long treatment with cytochalasin B, and negative in all other treatment schedules. In any case, no genotoxic compound would have been missed with schedules including a short and a long treatment time, whether the treatment was followed by a recovery period or not and whether cytochalasin B was used or not. Thus, these results show that CHL cells were suitable for accurately detecting clastogenic and aneugenic compounds of various types in the in vitro micronucleus test.     

Light-dependent polyploidy control by a CUE protein variant in Arabidopsis

Endoreduplication is a special cell cycle that increases ploidy without cell and nuclear division. In plants endoreduplication is essential for development. We isolated a dominant Arabidopsis mutant from activation tagging lines that had increased polyploidy in darkness. This mutant, ipd1-1D (increased polyploidy level in darkness 1-1D), shows longer hypocotyls and increased ploidy levels only in dark-grown seedlings. The corresponding gene encodes a protein that contains a CUE domain variant. IPD1 is specifically expressed in mitotically dividing cells. Furthermore we show that blue and far-red light can suppress the ploidy increase in ipd1-1D and also suppress the reporter expression in IPD1-promoter β-glucuronidase transgenic plants. These results suggest that IPD1 regulates the endocycle leading to hypocotyl elongation and this function is controlled by blue and far-red light. Electronic Supplementary Material Supplementary material is available for this article at Yuko Tsumoto and Takeshi Yoshizumi contributed equally to this work     

The effect of intracellular ppGpp levels on glutamate and lysine overproduction in Escherichia coli

Although the enhancement of amino-acid synthesis by guanosine-3',5'-tetraphosphate (ppGpp) is well known, the effect of intracellular ppGpp levels on amino-acid overproduction in Escherichia coli has not been investigated. In this study, we demonstrate that overexpression of the relA gene, encoding ppGpp synthetase, increases the accumulation of amino acids, such as glutamate and lysine, in amino-acid-overproducing strains of E. coli. Elevation of intracellular ppGpp levels due to depletion of required amino acids also enhances glutamate overproduction. Moreover, the extent of overproduction is highly dependent on the intracellular ppGpp level. These results demonstrate that amino-acid overproduction in E. coli is closely connected to amino-acid auxotrophy via the accumulation of ppGpp.     

Catalytic mechanism of ancestral L-lysine oxidase assigned by sequence data mining

A large number of protein sequences are registered in public databases such as PubMed. Functionally uncharacterized enzymes are included in these databases, some of which likely have potential for industrial applications. However, assignment of the enzymes remained difficult tasks for now. In this study, we assigned a total of 28 original sequences to uncharacterized enzymes in the FAD-dependent oxidase family expressed in some species of bacteria including Chryseobacterium, Flavobacterium, and Pedobactor. Progenitor sequence of the assigned 28 sequences was generated by ancestral sequence reconstruction, and the generated sequence exhibited L-lysine oxidase activity; thus, we named the enzyme AncLLysO. Crystal structures of ligand-free and ligand-bound forms of AncLLysO were determined, indicating that the enzyme recognizes L-Lys by hydrogen bond formation with R76 and E383. The binding of L-Lys to AncLLysO induced dynamic structural change at a plug loop formed by residues 251 to 254. Biochemical assays of AncLLysO variants revealed the functional importance of these substrate recognition residues and the plug loop. R76A and E383D variants were also observed to lose their activity, and the k_cat/K_m value of G251P and Y253A mutations were approximately 800- to 1800-fold lower than that of AncLLysO, despite the indirect interaction of the substrates with the mutated residues. Taken together, our data demonstrate that combinational approaches to sequence classification from database and ancestral sequence reconstruction may be effective not only to find new enzymes using databases of unknown sequences but also to elucidate their functions.     

Genetic divergence and evolutionary relationships in six species of genera Hoplobatrachus and Euphlyctis (Amphibia: Anura) from Bangladesh and other Asian countries revealed by mitochondrial gene sequences

To elucidate the species composition, genetic divergence, evolutionary relationships, and divergence time of Hoplobatrachus and Euphlyctis frogs (subfamily Dicroglossinae, family Ranidae) in Bangladesh and other Asian countries, we analyzed the mitochondrial Cyt b, 12S, and 16S rRNA genes of 252 specimens. Our phylogenetic analyses showed 13 major clades corresponding to several cryptic species as well as to nominal species in the two genera. The results suggested monophyly of Asian Hoplobatrachus species, but the position of African Hoplobatrachus occipitalis was not clarified. Nucleotide divergence and phylogenetic data suggested the presence of allopatric cryptic species allied to Euphlyctis hexadactylus in Sundarban, Bangladesh and several parapatric cryptic species in the Western Ghats, India. The presence of at least two allopatric cryptic species among diverged Euphlyctis cyanophlyctis in Bangladesh, India, and Sri Lanka was also suggested. In some cases, our estimated divergence times matched the paleogeological events of South and Southeast Asian regions that may have led to the divergence of Hoplobatrachus and Euphlyctis taxa. Especially, land formation at Bangladesh (15–10 Ma) may have allowed the spread of these frog taxa to Southeast Asian areas, and the aridification of central India (5.1–1.6 Ma) might have affected the gene flow of widely distributed species. The present study revealed prior underestimation of the richness of the amphibian fauna in this region, indicating the possible occurrence of many cryptic species among these groups.     

An amperometric biosensor based on a composite of single-walled carbon nanotubes, plasma-polymerized thin film, and an enzyme

We report on an amperometric biosensor that is based on a nanocomposite of carbon nanotubes (CNT), a nano-thin plasma-polymerized film (PPF), and glucose oxidase (GOx) as an enzyme model. A mixture of the GOx and a CNT film is sandwiched with 10-nm-thick acetonitrile PPFs. Under PPF layer was deposited onto a sputtered gold electrode. To facilitate the electrochemical communication between the CNT layer and GOx, CNT was treated with nitrogen or oxygen plasma. The resulting device showed that the oxidizing current response due to enzymatic reaction was 4-16-fold larger than that with only CNT or PPF, showing that the PPF and/or plasma process is an enzyme-friendly platform for designing electrochemical communication from the reaction center of GOx to the electrode via CNTs. The optimized glucose biosensor showed high sensitivity (sensitivity of 42 microA mM(-1)cm(-2), correlation coefficient of 0.992, linear response range of 0.025-2.2 mM, and a detection limit of 6 microM at signal/noise ratio of 3, +0.8 V versus Ag/AgCl), high selectivity (almost no interference by 0.5 mM ascorbic acid) for glucose quantification, and rapid response (<4 s to reach 95% of maximum response). Additionally, the devices showed a small and stable background current (0.35+/-0.013 microA) compared with the glucose response (ca. 10 microA at 10mM glucose) and suitable reproducibility from sample-to-sample (<3%, n=4).     

Quantitative telomeric overhang determination using a double-strand specific nuclease

Telomeres terminate in 3′ overhangs that function in end protection and the formation of t-loops. Determining the steps and factors involved in overhang processing is compromised by the inability to easily and accurately determine overhang size in the presence of many kilobases of double-stranded telomeric DNA. We here describe the use of a double-strand specific nuclease (DSN) that entirely digests double-stranded DNA including telomeres, leaving the overhangs intact so that they can be measured.