Functional engraftment of colon epithelium expanded in vitro from a single adult Lgr5⁺ stem cell

Adult stem-cell therapy holds promise for the treatment of gastrointestinal diseases. Here we describe methods for long-term expansion of colonic stem cells positive for leucine-rich repeat containing G protein-coupled receptor 5 (Lgr5(+) cells) in culture. To test the transplantability of these cells, we reintroduced cultured GFP(+) colon organoids into superficially damaged mouse colon. The transplanted donor cells readily integrated into the mouse colon, covering the area that lacked epithelium as a result of the introduced damage in recipient mice. At 4 weeks after transplantation, the donor-derived cells constituted a single-layered epithelium, which formed self-renewing crypts that were functionally and histologically normal. Moreover, we observed long-term (>6 months) engraftment with transplantation of organoids derived from a single Lgr5(+) colon stem cell after extensive in vitro expansion. These data show the feasibility of colon stem-cell therapy based on the in vitro expansion of a single adult colonic stem cell.     

Apoplastic plant subtilases support arbuscular mycorrhiza development in Lotus japonicus

In the arbuscular mycorrhiza (AM) symbiosis, plant roots accommodate Glomeromycota fungi within an intracellular compartment, the arbuscule. At this symbiotic interface, fungal hyphae are surrounded by a plant membrane, which creates an apoplastic compartment, the periarbuscular space (PAS) between fungal and plant cell. Despite the importance of the PAS for symbiotic signal and metabolite exchange, only few of its components have been identified. Here we show that two apoplastic plant proteases of the subtilase family are required for AM development. SbtM1 is the founder member of a family of arbuscular mycorrhiza-induced subtilase genes that occur in at least two clusters in the genome of the legume Lotus japonicus . A detailed expression analysis by RT-PCR revealed that SbtM1 , SbtM3 , SbtM4 and the more distantly related SbtS are all rapidly induced during development of arbuscular mycorrhiza, but only SbtS and SbtM4 are also up-regulated during root nodule symbiosis. Promoter–reporter fusions indicated specific activation in cells that are adjacent to intra-radical fungal hyphae or in cells that harbour them. Venus fluorescent protein was observed in the apoplast and the PAS when expressed from a fusion construct with the SbtM1 signal peptide or the full-length subtilase. Suppression of SbtM1 or SbtM3 by RNAi caused a decrease in intra-radical hyphae and arbuscule colonization, but had no effect on nodule formation. Our data indicate a role for these subtilases during the fungal infection process in particular arbuscule development.     

A chronosequence approach for detecting revegetation patterns after Sphagnum-peat mining, northern Japan

The aim of this study is to detect how vegetation development proceeds after Sphagnum-peat mining and how physical and chemical factors in groundwater are related to the revegetation patterns in Sarobetsu mire, Hokkaido, Japan. A total of 189 plots on peat-mining sites were set in a chronosequence and 18 plots were set on unmined control sites. A vegetation survey was conducted, and seasonal changes in groundwater levels and chemistry (pH, electrical conductance, total nitrogen, total phosphorus, anions, and cations) were monitored. Species richness and plot cover tended to increase with increasing age, but were significantly lower in mined sites than in unmined sites dominated by Sphagnum spp. The trends in vegetation change were (1) bare ground, (2) grasslands dominated by grasses and sedges, e.g., Rhyncohospora alba, Phragmites communis, and Moliniopsis japonica and (3) Sphagnum-dominated vegetation. The characteristics of groundwater level during the plant-growth period mostly determined vegetation recovery, i.e., Sphagnum establishment was promoted when groundwater declined greatly in early summer. The patterns of temporal vegetation changes affected by groundwater characteristics were detected by chronological sequence, and hydrological factors in groundwater were more important for revegetation than chemical factors. The original vegetation has not returned after three decades.     

Pharmacological Inhibition of Monoacylglycerol O-Acyltransferase 2 Improves Hyperlipidemia,Obesity, and Diabetes by Change in Intestinal Fat Utilization

Monoacylglycerol O-acyltransferase 2 (MGAT2) catalyzes the synthesis of diacylglycerol (DG), a triacylglycerol precursor and potential peripheral target for novel anti-obesity therapeutics. High-throughput screening identified lead compounds with MGAT2 inhibitory activity. Through structural modification, a potent, selective, and orally bioavailable MGAT2 inhibitor, compound A (compA), was discovered. CompA dose-dependently inhibited postprandial increases in plasma triglyceride (TG) levels. Metabolic flux analysis revealed that compA inhibited triglyceride/diacylglycerol resynthesis in the small intestine and increased free fatty acid and acyl-carnitine with shorter acyl chains than originally labelled fatty acid. CompA decreased high-fat diet (HFD) intake in C57BL/6J mice. MGAT2-null mice showed a similar phenotype as compA-treated mice and compA did not suppress a food intake in MGAT2 KO mice, indicating that the anorectic effects were dependent on MGAT2 inhibition. Chronic administration of compA significantly prevented body weight gain and fat accumulation in mice fed HFD. MGAT2 inhibition by CompA under severe diabetes ameliorated hyperglycemia and fatty liver in HFD-streptozotocin (STZ)-treated mice. Homeostatic model assessments (HOMA-IR) revealed that compA treatment significantly improved insulin sensitivity. The proximal half of the small intestine displayed weight gain following compA treatment. A similar phenomenon has been observed in Roux-en-Y gastric bypass-treated animals and some studies have reported that this intestinal remodeling is essential to the anti-diabetic effects of bariatric surgery. These results clearly demonstrated that MGAT2 inhibition improved dyslipidemia, obesity, and diabetes, suggesting that compA is an effective therapeutic for obesity-related metabolic disorders.     

Interspecific relations in forest floor coleopteron assemblages: Niche overlap and guild structure

In six different environments of the Towada-Hachimantai National Park, the forest floor coleopterons were sampled by means of pitfall traps with five different baits. Their niche breadths and niche overlaps were assessed for the electivity of habitat, bait, and habitat × bait, and compared with those expected from neutral models constructed randomly. The niche breadths in the observed assemblages were smaller than those expected from the neutral model, while the niche overlaps were more widely distributed than those expected from the neutral model. Mean overlaps between species neighbors in niche space, cluster analyses, and ordinations of species revealed a guild for habitat, bait and habitat × bait. Lawlor's (1979) gamma matrices concerning the habitat electivity show that the rates of mutualism resulted from indirect interspecific competitions are significantly smaller in the guild than in the whole assemblage. This suggests that the interspecific competition among the guild members is mitigated or even converted into mutualism by joining of the species which do not belong to the guild. The rates of mutualism were also significantly larger in the resource state of two dimensions (habitat × bait) than in that of either one. Therefore, if niche overlaps are measured on the basis of resource state of multi-dimensions, the rates of mutualism may increase further.     

Glucosylation of phenolic compounds by Datura innoxia suspension cultures

Datura innoxia grown in suspension cultures can glucosylate simple phenols. Three isomers of dihydroxybenzene (hydroquinone, resorcinol and catechol) were readily converted into their corresponding mono-β-glucosides. Both salicyl alcohol and salicylaldehyde fed to the cells were transformed specifically to isosalicin instead of salicin. Furthermore, the analysis of the cells treated with salicylic acid suggested the formation of its glucose ester in addition to the corresponding monoglucoside. Feeding experiments showed that the cultured cells possess a remarkably high capacity for glucosylation of hydroquinone, which was totally converted into arbutin within 10 hr after administration. The in vitro glucosylation of hydroquinone carried out by the cell-free extract demonstrated that this enzymic reaction requires the presence of UDPG as a high energy donor of glucose.     

Quantitative analysis of membrane components in a highly active O2-evolving photosystem II preparation from spinach chloroplasts

Stoichiometry of membrane components associated with Photosystem II was determined in a highly active O₂-evolving Photosystem II preparation isolated from spinach chloroplasts by the treatment with digitonin and Triton X-100. From the analysis with sodium dodecyl sulfate polyacrylamide gel electrophoresis and Triton X-114 phase partitioning, the preparation was shown to contain the reaction center protein (43 kDa), the light-harvesting chlorophyll-protein complex (the main band, 27 kDa), the herbicide-binding protein (32 kDa) and cytochrome b-559 (10 kDa) as hydrophobic proteins, and three proteins (33, 24 and 18 kDa) which probably constitute the O₂-evolution enzyme complex as hydrophilic proteins. These proteins were associated stoichiometrically with the Photosystem II reaction center: one Photosystem II reaction center, approx. 200 chlorophyll, one high-potential form of cytochrome b-559, one low-potential form of cytochrome b-559, one 33 kDa protein, one (to two) 24 kDa protein and one (to two) 18 kDa protein. Measurement of fluorescence induction showed the presence of three electron equivalents in the electron acceptor pool on the reducing side of Photosystem II in our preparation. Three molecules of plastoquinone A were detected per 200 chlorophyll molecules with high-performance liquid chromatography. The Photosystem II preparation contained four managanese atoms per 200 chlorophyll molecules.     

Crystal structure of VioE, a key player in the construction of the molecular skeleton of violacein

Violacein and the indolocarbazoles are naturally occurring bisindole products with various biological activities, including antitumor activity. Although these compounds have markedly different molecular skeletons, their biosynthetic pathways share the same intermediate "compound X," which is produced from L-tryptophan via indole-3-pyruvic acid imine. Compound X is a short-lived intermediate that is spontaneously converted to chromopyrrolic acid for indolocarbazole biosynthesis, whereas VioE transforms compound X into protodeoxyviolaceinic acid, which is further modified by other enzymes to produce violacein. Thus, VioE plays a key role in the construction of the molecular skeleton of violacein. Here, we present the crystal structure of VioE, which consists of two subunits, each of which forms a structure resembling a baseball glove. Each subunit has a positively charged pocket at the center of the concave surface of the structure. Mutagenesis analysis of the surface pocket and other surface residues showed that the surface pocket serves as an active site. We have also solved the crystal structure of a complex of VioE and phenylpyruvic acid as an analogue of a VioE-substrate complex. A docking simulation with VioE and the IPA imine dimer, which is proposed to be compound X, agreed with the results from the mutational analysis and the VioE-phenylpyruvic acid complex structure. Based on these results, we propose that VioE traps the highly reactive substrate within the surface pocket to suppress CPA formation and promote protodeoxyviolaceinic acid formation caused by proximity and orientation effects.