Identification and biochemical analysis of a homolog of a sulfate transporter from a vanadium-rich ascidian Ascidia sydneiensis samea

Background

Several species of ascidians accumulate extremely high levels of vanadium ions in the vacuoles of their blood cells (vanadocytes). The vacuoles of vanadocytes also contain many protons and sulfate ions. To maintain the concentration of sulfate ions, an active transporter must exist in the blood cells, but no such transporter has been reported in vanadium-accumulating ascidians.

Methods

We determined the concentration of vanadium and sulfate ions in the blood cells (except for the giant cells) of Ascidia sydneiensis samea. We cloned cDNA for an Slc13-type sulfate transporter, AsSUL1, expressed in the vanadocytes of A. sydneiensis samea. The synthetic mRNA of AsSUL1 was introduced into Xenopus oocytes, and its ability to transport sulfate ions was analyzed.

Results

The concentrations of vanadium and sulfate ions in the blood cells (except for the giant cells) were 38 mM and 86 mM, respectively. The concentration of sulfate ions in the blood plasma was 25 mM. The transport activity of AsSUL1 was dependent on sodium ions, and its maximum velocity and apparent affinity were 2500 pmol/oocyte/h and 1.75 mM, respectively.

General significance

This could account for active uptake of sulfate ions from blood plasma where sulfate concentration is 25 mM, as determined in this study.     

Detection and mapping of six miniF-encoded proteins by cloning analysis of dissected miniF segments

Summary Various DNA subfragments were derived from miniF DNA by complete or partial PstI cleavage, and cloned in the plasmid vectors pBR322 or dv1. The recombinant plasmids obtained were introduced into an Escherichia coli minicell-producing strain, and the plasmid-coded proteins were radiolabeled and analyzed by gel electrophoresis. Six miniF-encoded proteins, larger than 11 000 daltons, were detected and their coding regions were mapped on the F plasmid genome. Three of them were assigned by taking into account the known nucleotide sequences (Murotsu et al. 1981; K. Yoshioka, personal communication). The coding directions of some proteins were determined by inserting the lac promotor into one of the recombinant plasmids and analyzing the increase in production of the proteins. The coding direction of the five proteins analyzed so far was uniform. Comparison of these results with a functional map of miniF suggested possible roles of the proteins.     

Comparative studies of the haemagglutination of adult and umbilical cord erythrocytes by animal lectins

1. The sugar specificities of four lactose-binding lectins were studied through the agglutination of adult and/or umbilical cord human erythrocytes (AHRBC and/or CHRBC). 2. Rana catesbeiana egg lectin specifically agglutinated both intact blood group A-AHRBC and intact blood group A-CHRBC. 3. Rana catesbeiana liver lectin agglutinated intact A-AHRBC much more strongly than intact A-CHRBC. 4. Xenopus laevis skin lectin nonspecifically agglutinated AHRBC and CHRBC. 5. Plecoglossus altivelis egg lectin specifically agglutinated intact B-AHRBC, but weakly agglutinated intact B-CHRBC. 6. Comparative studies of lectin-induced AHRBC or CHRBC agglutination clarified the sugar-binding specificities of these lectins.     

Design and synthesis of 6-fluoro-2-naphthyl derivatives as novel CCR3 antagonists with reduced CYP2D6 inhibition

In our previous study on discovering novel types of CCR3 antagonists, we found a fluoronaphthalene derivative (1) that exhibited potent CCR3 inhibitory activity with an IC(50) value of 20 nM. However, compound 1 also inhibited human cytochrome P450 2D6 (CYP2D6) with an IC(50) value of 400 nM. In order to reduce its CYP2D6 inhibitory activity, we performed further systematic structural modifications on 1. In particular, we focused on reducing the number of lipophilic moieties in the biphenyl part of 1, using ClogD(7.4) values as the reference index of lipophilicity. This research led to the identification of N-{(3-exo)-8-[(6-fluoro-2-naphthyl)methyl]-8-azabicyclo[3.2.1]oct-3-yl}-3-(piperidin-1-ylcarbonyl)isonicotinamide 1-oxide (30) which showed comparable CCR3 inhibitory activity (IC(50)=23 nM) with much reduced CYP2D6 inhibitory activity (IC(50)=29,000 nM) compared with 1.     

Studies on the Metabolic Products of Oospora sp. (Oospora astringenes)

The chemical structure of oospolactone which is the metabolic product of Oospora astringenes was confirmed by the synthetical approach.     

Molecular nature of mutations induced by high-LET irradiation with argon and carbon ions in Arabidopsis thaliana

Linear energy transfer (LET) is an important parameter to be considered in heavy-ion mutagenesis. However, in plants, no quantitative data are available on the molecular nature of the mutations induced with high-LET radiation above 101-124keVμm(-1). In this study, we irradiated dry seeds of Arabidopsis thaliana with Ar and C ions with an LET of 290keVμm(-1). We analyzed the DNA alterations caused by the higher-LET radiation. Mutants were identified from the M(2) pools. In total, 14 and 13 mutated genes, including bin2, egy1, gl1, gl2, hy1, hy3-5, ttg1, and var2, were identified in the plants derived from Ar- and C-ions irradiation, respectively. In the mutants from both irradiations, deletion was the most frequent type of mutation; 13 of the 14 mutated genes from the Ar ion-irradiated plants and 11 of the 13 mutated genes from the C ion-irradiated plants harbored deletions. Analysis of junction regions generated by the 2 types of irradiation suggested that alternative non-homologous end-joining was the predominant pathway of repair of break points. Among the deletions, the proportion of large deletions (>100bp) was about 54% for Ar-ion irradiation and about 64% for C-ion irradiation. Both current results and previously reported data revealed that the proportions of the large deletions induced by 290-keVμm(-1) radiations were higher than those of the large deletions induced by lower-LET radiations (6% for 22.5-30.0keVμm(-1) and 27% for 101-124keVμm(-1)). Therefore, the 290keVμm(-1) heavy-ion beams can effectively induce large deletions and will prove useful as novel mutagens for plant breeding and analysis of gene functions, particularly tandemly arrayed genes.