Application of a single‐colony coculture technique to the isolation of hitherto unculturable gut bacteria

Molecular studies have led to postulation of a relationship between gut microbiota and certain diseases. However, because studies of hitherto uncultured species in vivo are essential for characterizing the biology and pathogenic properties of gut bacteria, techniques for culturing and isolating such bacteria must be developed. Here, a technique is described that partially overcomes the obstacles that prevent detection of interbacterial communication in vitro and are thus responsible for the failure to culture certain bacterial species. For this purpose, a ring with a membrane filter at the bottom was designed and a relatively simple nutrient medium was used instead of conventional media. Gut bacteria were cocultivated in soft agar separated by the membrane filter to simulate interbacterial communication in vitro. Use of this soft agar coculture technique led to the successful isolation of hitherto uncultured bacteria and the demonstration of multistage interbacterial communication among gut bacteria in vitro. Cultivation and isolation of single colonies of bacteria that require other bacteria for growth will enhance efforts to better understand the physiological and pathogenic roles of gut microbiota.     

KONJAC1 and 2 Are Key Factors for GDP-Mannose Generation and Affect l-Ascorbic Acid and Glucomannan Biosynthesis in Arabidopsis

Humans are unable to synthesize l-ascorbic acid (AsA), yet it is required as a cofactor in many critical biochemical reactions. The majority of human dietary AsA is obtained from plants. In Arabidopsis thaliana, a GDP-mannose pyrophosphorylase (GMPP), VITAMIN C DEFECTIVE1 (VTC1), catalyzes a rate-limiting step in AsA synthesis: the formation of GDP-Man. In this study, we identified two nucleotide sugar pyrophosphorylase-like proteins, KONJAC1 (KJC1) and KJC2, which stimulate the activity of VTC1. The kjc1kjc2 double mutant exhibited severe dwarfism, indicating that KJC proteins are important for growth and development. The kjc1 mutation reduced GMPP activity to 10% of wild-type levels, leading to a 60% reduction in AsA levels. On the contrary, overexpression of KJC1 significantly increased GMPP activity. The kjc1 and kjc1kjc2 mutants also exhibited significantly reduced levels of glucomannan, which is also synthesized from GDP-Man. Recombinant KJC1 and KJC2 enhanced the GMPP activity of recombinant VTC1 in vitro, while KJCs did not show GMPP activity. Yeast two-hybrid assays suggested that the stimulation of GMPP activity occurs via interaction of KJCs with VTC1. These results suggest that KJCs are key factors for the generation of GDP-Man and affect AsA level and glucomannan accumulation through the stimulation of VTC1 GMPP activity.     

In vitro protein import of a putative amino acid transporter from Arabidopsis thaliana into chloroplasts and its suborganellar localization

We identified a gene product of At5g19500 (At5g19500p) from Arabidopsis thaliana that is homologous to EcTyrP, a tyrosine-specific transporter from Escherichia coli. Computational analyses of the amino acid sequence of At5g19500p predicted 11 transmembrane domains (TMDs) and a potential plastid targeting signal at its amino terminus. As a first step toward understanding the possible role of At5g19500p in plant cells, we attempted to determine the localization of At5g19500p by an in vitro chloroplastic import assay using At5g19500p translated in a cell-free wheat germ system (Madin et al., Proc. Natl. Acad. Sci. USA, 97, 559-564 (2000)), followed by subfractionation of the chloroplasts. At5g19500p was successfully imported into chloroplasts, and the newly transported mature form of At5g19500p was recovered from the inner envelope membrane.     

A proposal of the method of deer density estimate without fecal decomposition rate: a case study of fecal accumulation rate technique in Japan

The accuracy of estimating deer density using the fecal pellet count method is greatly limited by variability of the fecal decomposition rate. The fecal accumulation rate technique can avoid the issue of decomposition rate. However, the precision of this technique is not clear when the decomposition rate is relatively high, such as in Japanese forests. We estimated deer population densities on Yakushima Island by the fecal accumulation rate technique and compared them between seasons. The estimated densities were similar to reported estimates, and did not differ seasonally, in accord with reports that deer on Yakushima do not migrate seasonally. Thus, we conclude that the fecal accumulation rate technique is applicable in Japanese forests.     

Alteration of ethanol tolerance caused by the deficiency in the genes associated with histone deacetylase complex in budding yeast

Upon exposure to 8% ethanol, survival and growth of yeast strains deficient in histone deacetylase complex genes was examined. Of the 18 mutants tested, the Δsir3 and Δsir4 strains showed higher resistance to ethanol, while the Δrco1, Δhos3, Δhda2, and Δhst1 strains were more sensitive than the wild type. Furthermore, these ethanol-resistant patterns varied under aerobic and anaerobic culture conditions.     

Lysophosphatidylinositol causes neurite retraction via GPR55, G13 and RhoA in PC12 cells

GPR55 was recently identified as a putative receptor for certain cannabinoids, and lysophosphatidylinositol (LPI). Recently, the role of cannabinoids as GPR55 agonists has been disputed by a number of reports, in part, because studies investigating GPR55 often utilized overexpression systems, such as the GPR55-overexpressing HEK293 cells, which make it difficult to deduce the physiological role of endogenous GPR55. In the present study, we found that PC12 cells, a neural model cell line, express endogenous GPR55, and by using these cells, we were able to examine the role of endogenous GPR55. Although GPR55 mRNA and protein were expressed in PC12 cells, neither CB(1) nor CB(2) mRNA was expressed in these cells. GPR55 was predominantly localized on the plasma membrane in undifferentiated PC12 cells. However, GPR55 was also localized in the growth cones or the ruffled border in differentiated PC12 cells, suggesting a potential role for GPR55 in the regulation of neurite elongation. LPI increased intracellular Ca(2+) concentration and RhoA activity, and induced ERK1/2 phosphorylation, whereas endogenous and synthetic cannabinoids did not, thereby suggesting that cannabinoids are not GPR55 agonists. LPI also caused neurite retraction in a time-dependent manner accompanied by the loss of neurofilament light chain and redistribution of actin in PC12 cells differentiated by NGF. This LPI-induced neurite retraction was found to be G(q)-independent and G(13)-dependent. Furthermore, inactivation of RhoA function via C3 toxin and GPR55 siRNA knockdown prevented LPI-induced neurite retraction. These results suggest that LPI, and not cannabinoids, causes neurite retraction in differentiated PC12 cells via a GPR55, G(13) and RhoA signaling pathway.     

Homeobox B3, FoxA1 and FoxA2 interactions in epithelial lung cell differentiation of the multipotent M3E3/C3 cell line

HOM/C homeobox (Hox) and forkhead box (Fox) factors are reported to be expressed in the foregut endoderm and are subsequently detected in a spatio-temporal pattern during lung development. Some of these factors were reported to influence the expression of lung marker proteins or to modulate lung development. To clarify the molecular mechanisms for generating functional lung cells from progenitor cell populations, we introduced the forkhead box factors, FoxA1 and FoxA2, and the homeobox factor, HoxB3, into the differentiation process in a multipotent hamster lung epithelial M3E3/C3 cell line. Ectopic expression of FoxA2 promoted differentiation to Clara-like cells with up-regulation of the expression of the lung marker proteins, Clara cell-specific 10-kDa protein and surfactant protein-B. In contrast, FoxA1 repressed the differentiation. HoxB3 transfection induced FoxA2 expression transiently at the pre-differentiation stage. The endogenous HoxB3 expression level decreased at later stages of Clara-like cell differentiation, and the attenuation was enhanced by FoxA2 transfection. HoxB3 is a putative upstream regulator that enhances FoxA2 expression at the pre-differentiation stage. In addition, we found that the expression of HoxA4, HoxA5, and HoxC9 increased differentially during Clara-like cell differentiation. These results suggest that HoxB3 may be a putative positive regulator of FoxA2 expression at the pre-differentiation stage, and those interactions of Fox factors and Hox factors could participate in Clara cell differentiation.