Two new species of <Emphasis Type="Italic">Zercon</Emphasis> (Acari: Zerconidae) from Turkey

Two new species of zerconid mites, Zercon uludagicus sp. n. and Z. huseyini sp. n., from Turkey are described and illustrated. The similarities and differences between the species are discussed. The lists of the taxonomic diagnostic key to adults of Zercon species known from Turkey are also given.     

Rad9 plays an important role in DNA mismatch repair through physical interaction with MLH1

Rad9 is conserved from yeast to humans and plays roles in DNA repair (homologous recombination repair, and base-pair excision repair) and cell cycle checkpoint controls. It has not previously been reported whether Rad9 is involved in DNA mismatch repair (MMR). In this study, we have demonstrated that both human and mouse Rad9 interacts physically with the MMR protein MLH1. Disruption of the interaction by a single-point mutation in Rad9 leads to significantly reduced MMR activity. This disruption does not affect S/M checkpoint control and the first round of G₂/M checkpoint control, nor does it alter cell sensitivity to UV light, gamma rays or hydroxyurea. Our data indicate that Rad9 is an important factor in MMR and carries out its MMR function specifically through interaction with MLH1.     

Water-soluble granules containing <Emphasis Type="Italic">Bacillus megaterium</Emphasis> for biological control of rice sheath blight: formulation,bacterial viability and efficacy testing

Endospores of B. megaterium were formulated in granule formulations with sodium alginate, lactose and polyvinylpyrrolidone (PVP K-30) by the wet granulation technique. The granule formulation exhibited good physical characteristics, such as high-water solubility and optimal viscosity, that would be suitable for spray application. The bacteria remained viable in the dry granule formulation at 10⁹ c.f.u./g after 24 months storage at room temperature. Under laboratory conditions, aqueous solutions of the formulation showed high activity against mycelial growth of R. solani (99.64 ± 0.14% mycelial inhibition). High viability of the bacterial antagonist on leaf sheath and leaf blade at day 7 after spraying with the formulation was observed (approximately 10⁶ c.f.u./g of plant). Application of an equivalent number of un-formulated endospores resulted in much loss of the bacterial endospores even 1 day after application. In a small pilot field study, an aqueous solution of the formulation (3%w/v) applied by spraying at days 1, 5 and 10 after pathogen inoculation of the rice plants was more effective in suppressing rice sheath blight disease than one application of a fungicide (Iprodione) at day 1. Additionally, rice plants sprayed with the aqueous solution of the granule formulation had higher panicle and whole kernel weights than those of fungicide-treated and control (untreated) plants.     

LC-MS/MS determination of helicid in human plasma and its application in pharmacokinetic studies

Helicid is a traditional Chinese medicine used to treat headache and insomnia with definite effects. To facilitate pharmacokinetic studies of helicid in man, a sensitive and specific LC-MS/MS method for the quantitative detection of helicid in human plasma was developed and validated. The method involved the addition of bergeninum as the internal standard (IS), protein precipitation, HPLC separation, and quantification by MS/MS system using negative electrospray ionization in the multiple reaction monitoring mode (MRM). The precursor→product ion transitions were monitored at m/z 282.8→120.9 for helicid and m/z 326.9→192.2 for the IS, respectively. The lower limit of quantification (LLOQ) was 0.2 μg/L. The calibration curves for helicid was linear over a concentration range of 0.2-20 μg/L. The intra- and inter-batch analyses of QC samples at 0.4, 2, 20 μg/L indicated good precision (%R.S.D. between 2.69 and 5.47%) and accuracy (between 96.15 and 105.05%). The helicid was stable in human plasma stored at room temperature for at least 24h, 4°C for at least 24h, -20°C for at least 1 month, and for routine three freeze-thaw cycles. This accurate and specific assay provides a useful method for evaluating the pharmacokinetic profile of helicid in humans.     

Self-organizing circuit assembly through spatiotemporally coordinated neuronal migration within geometric constraints

Background

Neurons are dynamically coupled with each other through neurite-mediated adhesion during development. Understanding the collective behavior of neurons in circuits is important for understanding neural development. While a number of genetic and activity-dependent factors regulating neuronal migration have been discovered on single cell level, systematic study of collective neuronal migration has been lacking. Various biological systems are shown to be self-organized, and it is not known if neural circuit assembly is self-organized. Besides, many of the molecular factors take effect through spatial patterns, and coupled biological systems exhibit emergent property in response to geometric constraints. How geometric constraints of the patterns regulate neuronal migration and circuit assembly of neurons within the patterns remains unexplored.

Methodology/Principal Findings

We established a two-dimensional model for studying collective neuronal migration of a circuit, with hippocampal neurons from embryonic rats on Matrigel-coated self-assembled monolayers (SAMs). When the neural circuit is subject to geometric constraints of a critical scale, we found that the collective behavior of neuronal migration is spatiotemporally coordinated. Neuronal somata that are evenly distributed upon adhesion tend to aggregate at the geometric center of the circuit, forming mono-clusters. Clustering formation is geometry-dependent, within a critical scale from 200 µm to approximately 500 µm. Finally, somata clustering is neuron-type specific, and glutamatergic and GABAergic neurons tend to aggregate homo-philically.

Conclusions/Significance

We demonstrate self-organization of neural circuits in response to geometric constraints through spatiotemporally coordinated neuronal migration, possibly via mechanical coupling. We found that such collective neuronal migration leads to somata clustering, and mono-cluster appears when the geometric constraints fall within a critical scale. The discovery of geometry-dependent collective neuronal migration and the formation of somata clustering in vitro shed light on neural development in vivo.     

Translation termination factor of eRFI of the ciliate Blepharisma japonicum recognizes all three stop codons

We have determined the type of stop codon specificity of Blepharisma japonicum translation termination factor eRF1 in an in vitro reconstituted eukaryotic translation system and in in vivo assay (the dual reporter system). We have shown that B. japonicum eRF1 retained specificity towards all three stop codons although efficiency of peptydyl-tRNA hydrolysis in the presence of UGA is reduced in an in vitro assay. We suggest that since the heterotrich B. japonicum represents the earliest diverged lineage on phylogenetic tree of ciliates, B. japonicum has the universal genetic code as ancestor group for all ciliates.     

Dissimilarity in the folding of human cytosolic creatine kinase isoenzymes

Creatine kinase (CK, EC 2.7.3.2) plays a key role in the energy homeostasis of excitable cells. The cytosolic human CK isoenzymes exist as homodimers (HMCK and HBCK) or a heterodimer (MBCK) formed by the muscle CK subunit (M) and/or brain CK subunit (B) with highly conserved three-dimensional structures composed of a small N-terminal domain (NTD) and a large C-terminal domain (CTD). The isoforms of CK provide a novel system to investigate the sequence/structural determinants of multimeric/multidomain protein folding. In this research, the role of NTD and CTD as well as the domain interactions in CK folding was investigated by comparing the equilibrium and kinetic folding parameters of HMCK, HBCK, MBCK and two domain-swapped chimeric forms (BnMc and MnBc). Spectroscopic results indicated that the five proteins had distinct structural features depending on the domain organizations. MBCK BnMc had the smallest CD signals and the lowest stability against guanidine chloride-induced denaturation. During the biphasic kinetic refolding, three proteins (HMCK, BnMc and MnBc), which contained either the NTD or CTD of the M subunit and similar microenvironments of the Trp fluorophores, refolded about 10-fold faster than HBCK for both the fast and slow phase. The fast folding of these three proteins led to an accumulation of the aggregation-prone intermediate and slowed down the reactivation rate thereby during the kinetic refolding. Our results suggested that the intra- and inter-subunit domain interactions modified the behavior of kinetic refolding. The alternation of domain interactions based on isoenzymes also provides a valuable strategy to improve the properties of multidomain enzymes in biotechnology.     

The Bryopsis hypnoides plastid genome: multimeric forms and complete nucleotide sequence

Background

Bryopsis hypnoides Lamouroux is a siphonous green alga, and its extruded protoplasm can aggregate spontaneously in seawater and develop into mature individuals. The chloroplast of B. hypnoides is the biggest organelle in the cell and shows strong autonomy. To better understand this organelle, we sequenced and analyzed the chloroplast genome of this green alga.

Principal Findings

A total of 111 functional genes, including 69 potential protein-coding genes, 5 ribosomal RNA genes, and 37 tRNA genes were identified. The genome size (153,429 bp), arrangement, and inverted-repeat (IR)-lacking structure of the B. hypnoides chloroplast DNA (cpDNA) closely resembles that of Chlorella vulgaris. Furthermore, our cytogenomic investigations using pulsed-field gel electrophoresis (PFGE) and southern blotting methods showed that the B. hypnoides cpDNA had multimeric forms, including monomer, dimer, trimer, tetramer, and even higher multimers, which is similar to the higher order organization observed previously for higher plant cpDNA. The relative amounts of the four multimeric cpDNA forms were estimated to be about 1, 1/2, 1/4, and 1/8 based on molecular hybridization analysis. Phylogenetic analyses based on a concatenated alignment of chloroplast protein sequences suggested that B. hypnoides is sister to all Chlorophyceae and this placement received moderate support.

Conclusion

All of the results suggest that the autonomy of the chloroplasts of B. hypnoides has little to do with the size and gene content of the cpDNA, and the IR-lacking structure of the chloroplasts indirectly demonstrated that the multimeric molecules might result from the random cleavage and fusion of replication intermediates instead of recombinational events.     

Isolation and characterization of a novel Ganoderma lucidum gene that differentially expressed between shaking culture and liquid static culture

Suppression subtractive hybridization (SSH) was preformed to investigate the differences of gene expression between the shaking culture mode and the liquid static culture mode which favors ganoderic acids production in Ganoderma lucidum. One novel gene preferentially expressed in liquid static culture was identified and analyzed. Its full length cDNA sequence and the 5′-flanking region were then obtained by rapid amplification of cDNA ends (RACE) and self-formed adaptor PCR (SEFA-PCR), respectively. Nucleotide sequence of the gene is not homologous to any of the known Ganoderma genes. The sequence analysis revealed that the open reading frame of this gene encodes a protein of 371 amino acids that has high homology with the mitogen-activated protein kinase (MAPK) of other five species-Postia (97%), Coprinopsis (91%), Neurospora (86%), Aspergillus (83%), and Saccharomyces (80%)-so that it can be defined as a G. lucidum MAPK gene (GenBank accession number: JF781125). Computer assisted analysis revealed that this new G. lucidum MAPK gene contains thirteen exons and twelve introns. The quantitative real-time RT-PCR (qRT-PCR) analysis showed that this new gene had a much higher expression level in liquid static culture than in traditional shaking culture. Results of this research established a good foundation for further study on the functions of the G. lucidum MAPK at the molecular level.