The atmospheric epiphyte Tillandsia ionantha is capable of surviving drought stress for 6 months or more without any exogenous water supply via an as of yet to be determined mechanism. When plants were soaked in water for 3 h, leaves absorbed a remarkably large amount of water (30-40% on the basis of fresh weight), exhibiting a bimodal absorption pattern. Radiolabeled water was taken up by the leaves by capillary action of the epidermal trichomes within 1 min (phase 1) and then transported intracellularly to leaf tissues over 3 h (phase 2). The removal of epidermal trichome wings from leaves as well as rinsing leaves with water significantly lowered the extracellular accumulation of water on leaf surfaces. The intracellular transport of water was inhibited by mercuric chloride, implicating the involvement of a water channel aquaporin in second-phase water absorption. Four cDNA clones (TiPIP1a, TiPIP1b, TiPIP1c, and TiPIP2a) homologous to PIP family aquaporins were isolated from the leaves, and RT-PCR showed that soaking plants in water stimulated the expression of TiPIP2a mRNA, suggesting the reinforcement in ability to rapidly absorb a large amount of water. The expression of TiPIP2a complementary RNA in Xenopus oocytes enhanced permeability, and treatment with inhibitors suggested that the water channel activity of TiPIP2a protein was regulated by phosphorylation. Thus, the high water uptake capability of T. ionantha leaves surviving drought is attributable to a bimodal trichome- and aquaporin-aided water uptake system based on rapid physical collection of water and subsequent, sustained chemical absorption.

Analyzing the intracellular contents and enzymatic activities of single cells is important for studying the physiological and pathological activities at the cellular level. For this purpose, we developed a simple single-cell lysis method by using a dense array of microwells of 10-30-pL volume fabricated by poly(dimethylsiloxane) (PDMS) and a commercially available cell lysis reagent. To demonstrate the performance of this single-cell lysis method, we carried out two different assays at the single-cell level: detection of proteins by antibody conjugated microbeads and measurement of protease activity by fluorescent substrates. The results indicated that this method readily enabled us to monitor protein levels and enzymatic activities in a single cell. Because this method required only an array of PDMS microwells and a fluorescence microscope, the simplicity of this platform opens a way to explore the biochemical characteristics of single cells even by those who are not familiar with microfluidic technology.

Plasmalogens are glycerophospholipids that contain a vinyl ether bond at the sn-1 position of glycerol backbone instead of an ester bond. Plasmalogens are indicated to have many important functions in mammalian cells. On the other hand, it is suggested that some gut microbiota plays many probiotic functions to human health. Presence of plasmalogens in Clostridium strains in gut microbiota is well-known, but presence of plasmalogens in Bifidobacterium longum (B. longum) strain, one of the most important probiotic gut microbiota, has not been reported. We identified plasmalogens in lipid extract from some B. longum species, but not from Bifidobacterium animalis (B. animalis) species which are another important strain of probiotic bifidobacteria. Major phospholipid classes of plasmalogens in B. longum species were cardiolipin, phosphatidylglycerol and phosphatidic acid. Almost all of the phospholipids from B. longum examined were indicated to be plasmalogens. Although major phospholipid classes of plasmalogens in human brain and major phospholipid classes of plasmalogens in B. longum are different, it is interesting to note that many reported functions of microbiota-gut-brain axis on human neurodegenerative diseases and those functions of plasmalogens on neurodegenerative diseases are overlapped. The presence of plasmalogens in B. longum species may play important roles for many probiotic effects of B. longum to human health.

Diet and exercise can alter the gut microbiota, but recent studies have assessed the impact of athletic competition on gut microbiota and host metabolites. We designed an open-label pilot study to investigate the effects of both official competition and a multi-strain lactic acid bacteria-fermented soymilk extract (LEX) on the gut microbiota in Japanese college endurance athletes. The analysis of fecal 16S rRNA metagenome and urinary metabolites was used to identify changes in gut microbiota composition and host metabolism. When the fecal microbiota were investigated before and after a race without using of a supplement (pre-observation period), there was an increase in the phylum Firmicutes and decrease in Bacteroidetes. However, no changes in these phyla were seen before and after a race in those who consumed LEX. Before and after LEX ingestion, changes in urinary metabolites included a significant reduction in yeast and fungal markers, neurotransmitters, and mitochondrial metabolites including the TCA cycle. There were several correlations between urinary metabolites and the composition of fecal microbiota. For example, the level of tricarballylic acid was positively correlated with the composition ratio of phylum Firmicutes (Pearson’s r = 0.66; p < 0.01). The bacterial species Parabacteroides distasonis was also found to correlate moderately with several urinary metabolites. These findings suggest two possibilities. First, endurance athletes experience significant fluctuations in gut microbiota after a single competition. Second, LEX ingestion may improve yeast and fungal overgrowth in the gastrointestinal tract and enhancing mitochondrial metabolic function.

We developed a new microscopic platform for the real-time analysis of molecular interactions by combining microbead-tagging techniques with total internal reflection fluorescent microscopy (TIRFM). The optical manipulation of probe microbeads, followed by photo immobilization on a solid surface, enabled us to generate arrays with extremely high density (>100 microbeads in a 25 µm × 25 µm area), and TIRFM made it possible to monitor the binding reactions of fluorescently labeled targets onto probe microbeads without removal of free targets. We demonstrated the high performance of this platform through analyses of interactions between antigen and antibody and between small compounds and proteins. Then, recombinant protein levels in total cellular lysates of Escherichia coli were quantified from the association kinetics using antibody-immobilized microbead arrays, which served as a model for a protein-profiling array. Furthermore, in combination with in vitro synthesis-coupled protein labeling, we could kinematically analyze the interaction of nuclear factor B (p50) with DNA. These results demonstrated that this platform enabled us to: (1) monitor binding processes of fluorescently labeled targets to multiple probes in real-time without removal of free targets, (2) determine concentrations of free targets only from the association kinetics at an early phase, and (3) greatly reduce the required volume of the target solution, in principle to subnanoliter, for molecular interaction analysis. The unique features of this microbead-based microarray system open the way to explore molecular interactions with a wide range of affinities in extremely small volumes of target solutions, such as extracts from single cells.

No abstract

No abstract

No abstract