NoxA activation by the small GTPase RacA is required to maintain a mutualistic symbiotic association between Epichloë festucae and perennial ryegrass

Small GTPases of the Rac group play a key regulatory role in NADPH oxidase catalysed production of reactive oxygen species (ROS) in mammals and plants, but very little evidence is available for a corresponding role in fungi. We recently showed that ROS produced by a specific fungal NADPH oxidase isoform, NoxA, are crucial in regulating hyphal morphogenesis and growth in the mutualistic symbiotic interaction between Epichloë festucae and perennial ryegrass. We demonstrate here that E. festucae RacA is required for NoxA activation and regulated production of ROS to maintain a symbiotic interaction. Deletion of racA resulted in decreased ROS production, reduction of radial growth and hyper‐branching of the hyphae in culture. In contrast, in planta the racA mutant showed extensive colonization of the host plant, resulting in stunting and precocious senescence of the host plants. Strains expressing a dominant active (DA) allele of RacA had increased ROS production, increased aerial hyphae and reduced radial growth. These results demonstrate that RacA plays a crucial role in regulating ROS production by NoxA, in order to control hyphal morphogenesis and growth of the endophyte in planta.     

Triplex formation with 2'-O,4'-C-ethylene-bridged nucleic acids (ENA) having C3'-endo conformation at physiological pH

Antigenes, which are substances that inhibit gene expression by binding to double-stranded DNA (dsDNA) in a sequence-specific manner, are currently sought for the treatment of various gene-related diseases. As such antigenes, we developed new nuclease-resistant oligopyrimidine nucleotides that are partially modified with 2′-O,4′-C-ethylene nucleic acids (ENA), which are constrained in the C3′-endo conformation and can form a triplex with dsDNA at physiological pH. It was found that these oligonucleotides formed triplexes similarly to those partially modified with 2′-O,4′-C-methylene nucleic acids (2′,4′-BNA or LNA), as determined by UV melting analyses, electromobility shift assays, CD spectral analyses and restriction enzyme inhibition assays. In our studies, oligonucleotides fully modified with ENA have δ torsion angle values that are marginally higher than those of 2′,4′-BNA/LNA. ENA oligonucleotides present in 10-fold the amount of dsDNA were found to be favorable in forming triplexes. These results provide useful information for the future design of triplex-forming oligonucleotides fully modified with such nucleic acids constrained in the C3′-endo conformation considering that oligonucleotides fully modified with 2′,4′-BNA/LNA do not form triplexes.     

Chemical knockdown of protein-tyrosine phosphatase 1B by 1,2-naphthoquinone through covalent modification causes persistent transactivation of epidermal growth factor receptor

1,2-Naphthoquinone (1,2-NQ), an atmospheric contaminant, causes the contraction of guinea pig trachea through the activation of epidermal growth factor receptor (EGFR) by inhibiting protein-tyrosine phosphatases (PTPs). Phosphorylation of EGFR is negatively regulated by PTPs, but details of the mechanism by which 1,2-NQ inhibits PTPs have not been elucidated. Results described in this report demonstrate that 1,2-NQ forms covalent bonds with PTP1B after exposure to human epithelial A431 cells. In this study, a concentration-dependent phosphorylation of EGFR was found to be coupled to the reduction of PTP activity in the cells. The reduction in PTP activity was due to the irreversible modification of PTP1B, and when PTP1B was overexpressed by the cells, the 1,2-NQ-mediated EGFR phosphorylation was suppressed. Studies with purified PTP1B and 1,2-NQ showed that the reduction in enzyme activity was due to a nucleophilic attack by the quinone on the enzyme, to form covalent bonds. Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry analysis and mutation experiments revealed that PTP1B inactivation was primarily due to covalent attachment of the quinone to Cys-121 of the enzyme, with binding to His-25 and Cys-215 as well. Collectively, the results show that covalent attachment of 1,2-NQ to PTP1B is at least partially responsible for the reduction of PTP activity, which leads to prolonged transactivation of EGFR in the cells.     

Numerically evaluated functional equivalence between chaotic dynamics in neural networks and cellular automata under totalistic rules

Chaotic dynamics in a recurrent neural network model and in two-dimensional cellular automata, where both have finite but large degrees of freedom, are investigated from the viewpoint of harnessing chaos and are applied to motion control to indicate that both have potential capabilities for complex function control by simple rule(s). An important point is that chaotic dynamics generated in these two systems give us autonomous complex pattern dynamics itinerating through intermediate state points between embedded patterns (attractors) in high-dimensional state space. An application of these chaotic dynamics to complex controlling is proposed based on an idea that with the use of simple adaptive switching between a weakly chaotic regime and a strongly chaotic regime, complex problems can be solved. As an actual example, a two-dimensional maze, where it should be noted that the spatial structure of the maze is one of typical ill-posed problems, is solved with the use of chaos in both systems. Our computer simulations show that the success rate over 300 trials is much better, at least, than that of a random number generator. Our functional simulations indicate that both systems are almost equivalent from the viewpoint of functional aspects based on our idea, harnessing of chaos.     

Erp1/Emi2 is essential for the meiosis I to meiosis II transition in Xenopus oocytes

Erp1 (also called Emi2), an inhibitor of the APC/C ubiquitin ligase, is a key component of cytostatic factor (CSF) responsible for Meta-II arrest in vertebrate eggs. Reportedly, however, Erp1 is expressed even during meiosis I in Xenopus oocytes. If so, it is a puzzle why normally maturing oocytes cannot arrest at Meta-I. Here, we show that actually Erp1 synthesis begins only around the end of meiosis I in Xenopus oocytes, and that specific inhibition of Erp1 synthesis by morpholino oligos prevents entry into meiosis II. Furthermore, we demonstrate that premature, ectopic expression of Erp1 at physiological Meta-II levels can arrest maturing oocytes at Meta-I. Thus, our results show the essential role for Erp1 in the meiosis I/meiosis II transition in Xenopus oocytes and can explain why normally maturing oocytes cannot arrest at Meta-I.     

Determination of metabolic flux changes during fed-batch cultivation from measurements of intracellular amino acids by LC-MS/MS

Metabolic flux analysis using (13)C-labeled substrates is a well-developed method for investigating cellular behavior in steady-state culture condition. To extend its application, in particular to typical industrial conditions, such as batch and fed-batch cultivations, a novel method of (13)C metabolic flux analysis is proposed. An isotopomer balancing model was developed to elucidate flux distributions in the central metabolism and all amino acids synthetic pathways. A lysine-producing strain of Escherichia coli was cultivated by fed-batch mode in a growth medium containing yeast extract. Mass distribution data was derived from both intracellular free amino acids and proteinogenic amino acids measured by LC-MS/MS, and a correction parameter for the protein turnover effect on the mass distributions of intracellular amino acids was introduced. Metabolic flux distributions were determined in both exponential and stationary phases. Using this new approach, a culture phase-dependent metabolic shift was detected in the fed-batch culture. The approach presented here has great potential for investigating cellular behavior in industrial processes, independent of cultivation modes, metabolic phase and growth medium.     

Molecular hydrogen suppresses Porphyromonas gingivalis lipopolysaccharide-induced increases in interleukin-1 alpha and interleukin-6 secretion in human gingival cells

Molecular and Cellular Biochemistry - Periodontitis is defined as a multifactorial polymicrobial infection accompanied by inflammatory reactions. Porphyromonas gingivalis (Pg) is known as a major...     

Renadirsen,a Novel 2′OMeRNA/ENA® Chimera Antisense Oligonucleotide,Induces Robust Exon 45 Skipping for Dystrophin In Vivo

Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease caused by out-of-frame or nonsense mutation in the dystrophin gene. It begins with a loss of ambulation between 9 and 14 years of age, followed by various other symptoms including cardiac dysfunction. Exon skipping of patients’ DMD pre-mRNA induced by antisense oligonucleotides (AOs) is expected to produce shorter but partly functional dystrophin proteins, such as those possessed by patients with the less severe Becker muscular dystrophy. We are working on developing modified nucleotides, such as 2′-O,4′-C-ethylene-bridged nucleic acids (ENAs), possessing high nuclease resistance and high affinity for complementary RNA strands. Here, we demonstrate the preclinical characteristics (exon-skipping activity in vivo, stability in blood, pharmacokinetics, and tissue distribution) of renadirsen, a novel AO modified with 2′-O-methyl RNA/ENA chimera phosphorothioate designed for dystrophin exon 45 skipping and currently under clinical trials. Notably, systemic delivery of renadirsen sodium promoted dystrophin exon skipping in cardiac muscle, skeletal muscle, and diaphragm, compared with AOs with the same sequence as renadirsen but conventionally modified by PMO and 2′OMePS. These findings suggest the promise of renadirsen sodium as a therapeutic agent that improves not only skeletal muscle symptoms but also other symptoms in DMD patients, such as cardiac dysfunction.     

The effect of entomopathogenic Lecanicillium spp. (Hypocreales: Cordycipitaceae) on the aphid parasitoid Aphidius colemani (Hymenoptera: Aphidiinae)

The effects of entomopathogenic Lecanicillium spp. (Zare and Gams) on Aphidius colemani (Viereck) adult longevity and ovipositional behavior and on the emergence rate and longevity of the F₁ generation of this parasitoid insect were evaluated under laboratory conditions. Moreover, the ability of three strains of Lecanicillium spp. to penetrate the cuticle of aphids parasitized by A. colemani at different larval stages was investigated. Although fungal treatments at high concentrations (10⁷?conidia/ml) reduced adult longevity, no significant effects were observed at lower concentrations. The number of ovipositional probings, sting failure rate of the ovipositor, and time intervals between periods of ovipositional behavior were not significantly different between the experimental groups and controls at all concentrations for all fungal strains. Fungal inoculation showed no significant effects on the emergence rate and longevity of the adult A. colemani F₁ generation. Fungal penetration was detected in 60?% of aphids inoculated with fungi 7?days after parasitoid exposure. However, fungal detection was drastically reduced and undetectable when fungal inoculation occurred 8 and 9?days after parasitization, respectively. Our results suggest the coapplication of Lecanicillium spp. and A. colemani for aphid control is possible under certain conditions because of their high compatibility.