Autotaxin Overexpression Causes Embryonic Lethality and Vascular Defects

Autotaxin (ATX) is a secretory protein, which converts lysophospholipids to lysophosphatidic acid (LPA), and is essential for embryonic vascular formation. ATX is abundantly detected in various biological fluids and its level is elevated in some pathophysiological conditions. However, the roles of elevated ATX levels remain to be elucidated. In this study, we generated conditional transgenic (Tg) mice overexpressing ATX and examined the effects of excess LPA signalling. We found that ATX overexpression in the embryonic period caused severe vascular defects and was lethal around E9.5. ATX was conditionally overexpressed in the neonatal period using the Cre/loxP system, which resulted in a marked increase in the plasma LPA level. This resulted in retinal vascular defects including abnormal vascular plexus and increased vascular regression. Our findings indicate that the ATX level must be carefully regulated to ensure coordinated vascular formation     

Efficient generation of genome-modified mice via offset-nicking by CRISPR/Cas system

The mammalian zygote-mediated CRISPR/Cas system can efficiently generate targeted genome-modified animals. However, this system is limited by the risk of off-target mutations. Here we show that offset-nicking by Cas9 nickase and paired gRNAs allows us to generate region deleted mice and targeted knock-in mice without off-target mutations.     

Effects of sedge and cottongrass tussocks on plant establishment patterns in a post-mined peatland,northern Japan

Facilitation (positive inter-specific interaction) plays an important role in promoting succession in harsh environments. To examine whether tussocks facilitate the establishment of other species, after peat mining, investigations were carried out in a formerly Sphagnum-dominated wetland (Sarobetsu mire, northern Japan). Two tussock-forming species, Carex middendorffii and Eriophorum vaginatum, have established in sparsely vegetated areas, with a dry ground surface, since peat extraction ended. The following factors were examined, in three microhabitats created by tussocks (center = raised tussock center, edge = tussock edge covered with litter, and flat = flat areas without tussocks): (1) relationships between tussock microhabitats and plant distributions, and (2) the effects of tussocks on survival, growth, flowering and seed immigration of common species. Two (1 × 10 m) plots were established, in each of three sparsely vegetated sites, in September 2005. Tussocks were mapped in each plot, and species, location, flowering, growth stage (seedling, juvenile and fertile) and size of all plants were recorded, during snow-free periods from September 2005 to September 2006. Seed traps were used to investigate seed dispersal from June to October 2006. Four native species, Drosera rotundifolia, Lobelia sessilifolia, Moliniopsis japonica, Solidago virgaurea, and an exotic species, Hypochaeris radicata, were most common. During seedling and juvenile stages, these species were distributed more densely at the tussock edge than in the flat areas, but were less common at the center. H. radicata had a higher survival rate at the edge than in the flat during the winter. The annual growth of H. radicata, L. sessilifolia and S. virgaurea was higher at the edge. Seed traps detected that D. rotundifolia seeds accumulate more at the edge. In conclusion, tussocks facilitated plant establishment in the edge microhabitat by providing litter cover, enhancing seed accumulation, germination and survival, and thus promoted revegetation. However, Sphagnum mosses have not established in the study sites, and the vegetation differs strongly from the areas where no peat mining had taken place.     

Ploidy-specific interactions of three host plants with arbuscular mycorrhizal fungi: Does genome copy number matter?

? Premise of the study: Polyploidy has been shown to affect different plant traits and modulate interactions between plants and other organisms, such as pollinators and herbivores. However, no information is available on whether it can also shape the functioning of mycorrhizal symbiosis. ? Methods: The mycorrhizal growth response was assessed for three angiosperms with intraspecific ploidy variation. Different cytotypes of Aster amellus, Campanula gentilis, and Pimpinella saxifraga were either left uninoculated or were inoculated with arbuscular mycorrhizal (AM) fungi in a pot experiment. After 3 mo of cultivation in a greenhouse, plant growth, phosphorus concentration in the shoot biomass, and development of the AM symbiosis were evaluated. ? Key results: No significant ploidy-specific differences in AM development were recorded. The inoculation led to consistently greater phosphorus uptake; however, the effect on plant growth differed considerably among plant species, populations, ploidy levels, and AM species. A salient ploidy-specific response was observed in A. amellus. Whereas diploid plants benefited from AM inoculation, the hexaploids consistently showed negative or no-growth responses (depending on the AM species). In contrast to A. amellus, no interactions between inoculation and ploidy were observed in C. gentilis and P. saxifraga. ? Conclusions: The first evidence is provided of a ploidy-specific response of a mycotrophic plant to AM fungi. Our results demonstrate the complexity of interaction between plants and associated AM fungi, with the ploidy level of the host plant being one component that may modulate the functioning of the symbiosis.     

Functional Characterization of Corynebacterium alkanolyticum β-Xylosidase and Xyloside ABC Transporter in Corynebacterium glutamicum

The Corynebacterium alkanolyticum xylEFGD gene cluster comprises the xylD gene that encodes an intracellular β-xylosidase next to the xylEFG operon encoding a substrate-binding protein and two membrane permease proteins of a xyloside ABC transporter. Cloning of the cluster revealed a recombinant β-xylosidase of moderately high activity (turnover for p-nitrophenyl-β-d-xylopyranoside of 111 ± 4 s⁻¹), weak α-l-arabinofuranosidase activity (turnover for p-nitrophenyl-α-l-arabinofuranoside of 5 ± 1 s⁻¹), and high tolerance to product inhibition (K_i for xylose of 67.6 ± 2.6 mM). Heterologous expression of the entire cluster under the control of the strong constitutive tac promoter in the Corynebacterium glutamicum xylose-fermenting strain X1 enabled the resultant strain X1EFGD to rapidly utilize not only xylooligosaccharides but also arabino-xylooligosaccharides. The ability to utilize arabino-xylooligosaccharides depended on cgR_2369, a gene encoding a multitask ATP-binding protein. Heterologous expression of the contiguous xylD gene in strain X1 led to strain X1D with 10-fold greater β-xylosidase activity than strain X1EFGD, albeit with a total loss of arabino-xylooligosaccharide utilization ability and only half the ability to utilize xylooligosaccharides. The findings suggest some inherent ability of C. glutamicum to take up xylooligosaccharides, an ability that is enhanced by in the presence of a functional xylEFG-encoded xyloside ABC transporter. The finding that xylEFG imparts nonnative ability to take up arabino-xylooligosaccharides should be useful in constructing industrial strains with efficient fermentation of arabinoxylan, a major component of lignocellulosic biomass hydrolysates.     

Ameliorative effects of mulberry (Morus alba L.) leaves on hyperlipidemia in rats fed a high-fat diet: induction of fatty acid oxidation, inhibition of lipogenesis, and suppression of oxidative stress

To determine the effects of mulberry (Morus alba L.) leaves on hyperlipidemia, we performed gene expression profiling of the liver. Rats were fed a high-fat diet and administered mulberry leaves for 7 weeks. Plasma triglyceride and non-esterified fatty acid levels were significantly lower in the rats treated with mulberry leaves as compared with the untreated rats. DNA microarray analysis revealed that mulberry leaves upregulated expression of the genes involved in α-, β- and ω-oxidation of fatty acids, mainly related to the peroxisome proliferator-activated receptor signaling pathway, and downregulated the genes involved in lipogenesis. Furthermore, treatment with mulberry leaves upregulated expression of the genes involved in the response to oxidative stress. These results indicate that consumption of fatty acids and inhibition of lipogenesis are responsible for the reduction in plasma lipids caused by mulberry administration. In addition, mulberry treatment maintains the body's oxidative state at a low level despite enhancing fatty acid oxidation.     

Phylogeography of Pulsatilla cernua (Ranunculaceae), a grassland species,in Japan

The genetic diversity and structure of Pulsatilla cernua, a continental‐grassland relict, were investigated using variations in chloroplast DNA (cpDNA) and microsatellites of nuclear DNA. In the analyses of three cpDNA regions, 17 haplotypes were found in 24 populations of P. cernua from Japan, Korea, and Russia. Although the route and time of migration between the continent of Asia and Japan could not be well resolved, the cpDNA haplotype network suggests the existence of several ancient lineages in Japan and a recent secondary migration from Japan to the continent. Microsatellite analyses did not indicate genetic structure among the Japanese populations, indicating the existence of gene flow across the distribution area until recently. These results indicate that the present fragmentation of P. cernua in Japan may reflect a rapid, recent reduction from a previously large, continuous distribution.     

ASC-mediated NF-kappaB activation leading to interleukin-8 production requires caspase-8 and is inhibited by CLARP

ASC is an adaptor molecule that mediates apoptotic and inflammatory signals from several Apaf-1-like molecules, including CARD12/Ipaf, cryopyrin/PYPAF1, PYPAF5, PYPAF7, and NALP1. To characterize the signaling pathway mediated by ASC, we established cell lines in which muramyl dipeptide, the bacterial component recognized by another Apaf-1-like molecule, Nod2, induced an interaction between a CARD12-Nod2 chimeric protein and ASC, and elicited cell autonomous NF-kappaB activation. This response required caspase-8, and was suppressed by CLARP/FLIP, an inhibitor of caspase-8. The catalytic activity of caspase-8 was required for the ASC-mediated NF-kappaB activation when caspase-8 was expressed at an endogenous level, although it was not essential when caspase-8 was overexpressed. In contrast, FADD, the adaptor protein linking Fas and caspase-8, was not required for this response. Consistently, ASC recruited caspase-8 and CLARP but not FADD and Nod2 to its speck-like aggregates in cells. Finally, muramyl dipeptide induced interleukin-8 production in MAIL8 cells. These results are the first to indicate that caspase-8 plays an important role in the ASC-mediated NF-kappaB activation, and that the ASC-mediated NF-kappaB activation actually induces physiologically relevant gene expression.