The effect of claudin-15 deletion on cationic selectivity and transport in paracellular pathways of the cecum and large intestine.

The large intestine plays a pivotal role in water and electrolyte balance. Paracellular transport may play a role in ion transport mechanisms in the cecum and large intestine; however, these molecular mechanisms and their physiological roles have not been fully studied. Claudin-15 forms a cation channel in tight junctions in the small intestine, but its role in the cecum and large intestine has not been investigated. This study aimed to explore the physiological role of claudin-15 in the cecum and large intestine using claudin-15 (Cldn15) KO mice. Electrical conductance, short-circuit current, Na+ flux, and dilution potential were assessed in isolated tissue preparations mounted in Ussing chambers. The induced short-circuit current of short-chain fatty acids, which are fermentative products in the intestinal tract, was also measured. Compared to wild type mice, the electrical conductance and paracellular Na+ flux was decreased in the cecum, but not the middle large intestine, while in both the cecum and the middle large intestine, paracellular Na+ permeability was decreased in Cldn15 KO mice. These results suggest that claudin-15 is responsible for Na+ permeability in the tight junctions of the cecum and large intestine and decreased Na+ permeability in the cecum may cause impaired absorption function.

Na+-dependent intestinal glucose absorption mechanisms and its luminal Na+ homeostasis across metamorphosis from tadpoles to frogs.

The abrupt morphological changes of the intestine during metamorphosis have been detailed in frogs. The features of intestinal metamorphosis are shortening of the intestine and remodeling of the intestinal epithelium. It is believed that the purpose of the morphological changes of the intestine is adaptation from aquatic herbivorous to carnivorous life. However, little is known about the physiological importance of these morphological changes. To elucidate the functional changes during metamorphosis, we measured luminal Na+ concentrations and Na+-dependent glucose uptake in tadpoles and adult African clawed frogs Xenopus laevis. The small intestine was isolated and divided into four segments in length, the luminal contents collected for analysis of ion concentration by ion chromatography. Phlorizin-sensitive glucose-induced short-circuit current (ΔIsc) was measured in intestinal preparations mounted in Ussing chambers. Although dietary sodium intake was extremely low in tadpoles, luminal Na+ concentration gradually increased along the proximal to the middle part of the intestine (>70 mM), and this Na+ concentration was comparable with that of carnivorous adult frogs. The increment of glucose-induced ΔIsc was observed in tadpole intestine. We also measured the ΔIsc induced by acetic acid, which is the major short-chain fatty acid produced by fermentation. The expression levels of mRNA for Na+-dependent glucose transporter 1 and tight junction protein claudin-15 in each intestinal segment was measured. These results suggest that luminal Na+ homeostasis is important and luminal Na+ is kept at a high concentration for Na+-dependent nutrient absorption mechanisms.

The major route for chlorophyll synthesis includes [3,8-divinyl]-chlorophyllide a reduction in Arabidopsis thaliana.

In most reviews on Chl biosynthesis, Chl is described as being synthesized via the route involving the reduction of [3,8-divinyl]-protochlorophyllide a. However, the possibility remains that the conversion of the divinyl form of the Chl intermediate to its monovinyl form takes place at other enzymatic steps. To determine the actual route of Chl biosynthesis, we examined the substrate specificity of the formerly named [3,8-divinyl]-protochlorophyllide a 8-vinyl reductase (DVR) in vitro. In addition, we investigated the accumulation of various Chl intermediates in etiolated seedlings in vivo. Collectively, these studies indicate that [3,8-divinyl]-chlorophyllide a is the major substrate of DVR.

The N-terminal domain of chlorophyllide a oxygenase confers protein instability in response to chlorophyll B accumulation in Arabidopsis.

Plants acclimate to variations in light intensity by changing the antenna size of photosystems. This acclimation allows them to undergo efficient photosynthesis and creates a protective strategy to minimize photodamage. Chlorophyll b synthesis by chlorophyllide a oxygenase (CAO) is a key regulatory step in the control of antenna size. Recently, we found that higher plant CAOs consist of three domains (A, B, and C domains) and confirmed that the C domain possesses catalytic function. To investigate the function of the A domain, we fused various combinations of these three domains with green fluorescent protein (GFP) and introduced them into Arabidopsis thaliana. When a full-length CAO-GFP fusion protein was introduced into a chlorophyll b-less chlorina1-1 mutant, chlorophyll b accumulated to almost the same levels as in the chlorophyll b-containing Columbia wild type, but the CAO-GFP could not be detected by immunoblotting. By contrast, when a GFP-C domain fusion was introduced into chlorina1-1 or Columbia wild type, a large amount of GFP-C domain protein accumulated and the chlorophyll a/b ratio decreased drastically from 3.6 to 2.2 in Columbia wild type. When an A domain-GFP was introduced into Columbia wild type, A domain-GFP levels were very low. Conversely, a large amount of the protein accumulated when it was introduced into the chlorina1-1 mutant. These results indicate that the A domain may sense the presence of chlorophyll b and regulate the accumulation of CAO protein in the chloroplasts.

Identification of a vinyl reductase gene for chlorophyll synthesis in Arabidopsis thaliana and implications for the evolution of Prochlorococcus species.

Chlorophyll metabolism has been extensively studied with various organisms, and almost all of the chlorophyll biosynthetic genes have been identified in higher plants. However, only the gene for 3,8-divinyl protochlorophyllide a 8-vinyl reductase (DVR), which is indispensable for monovinyl chlorophyll synthesis, has not been identified yet. In this study, we isolated an Arabidopsis thaliana mutant that accumulated divinyl chlorophyll instead of monovinyl chlorophyll by ethyl methanesulfonate mutagenesis. Map-based cloning of this mutant resulted in the identification of a gene (AT5G18660) that shows sequence similarity with isoflavone reductase genes. The mutant phenotype was complemented by the transformation with the wild-type gene. A recombinant protein encoded by AT5G18660 was expressed in Escherichia coli and found to catalyze the conversion of divinyl chlorophyllide to monovinyl chlorophyllide, thereby demonstrating that the gene encodes a functional DVR. DVR is encoded by a single copy gene in the A. thaliana genome. With the identification of DVR, finally all genes required for chlorophyll biosynthesis have been identified in higher plants. Analysis of the complete genome of A. thaliana showed that it has 15 enzymes encoded by 27 genes for chlorophyll biosynthesis from glutamyl-tRNA(glu) to chlorophyll b. Furthermore, identification of the DVR gene helped understanding the evolution of Prochlorococcus marinus, a marine cyanobacterium that is dominant in the open ocean and is uncommon in using divinyl chlorophylls. A DVR homolog was not found in the genome of P. marinus but found in the Synechococcus sp WH8102 genome, which is consistent with the distribution of divinyl chlorophyll in marine cyanobacteria of the genera Prochlorococcus and Synechococcus.

Specificity of one-base mismatch detection with MagiProbe.

MagiProbe have been demonstrated to be a homogeneous type of probe that emits fluorescence upon hybridization and with specificity for one-base mismatch. To obtain more comprehensive information for the properties of mismatch recognition, examination of mismatch recognition of MagiProbe for the eight single-base mismatches in variety of sequence contexts was performed. The results indicate that the recognition of base mismatches is influenced predominantly by combination of mispaired bases.