Kosuke Shimogawara

Establishment of publicly available cDNA material and information resource of Chlamydomonas reinhardtii (Chlorophyta) to facilitate gene function analysis

Abstract A large-scale analysis of expressed sequence tags (EST) was conducted in a unicellular green alga, Chlamydomonas reinhardtii. To complement the previously published EST sequences obtained from photoautotrophically grown cells and cells under CO2 stress condition, a mixture of cells grown under various stress conditions including light, temperature, salt and limited nutrient was used as a material to construct the complementary DNA library. We have generated 12,842 5′ end sequences from this library, making the collection 50,832 in its entirety. To speed up the data processing step, an internal semiautomatic operation pipeline was constructed. Using this system, quality check of the generated sequences, trimming, and database search for primary annotation was performed. Clustering of the EST sequences by BLAST followed by Phrap has produced 3748 consensus sequences and 7721 singletons. To make this high-quality set of assemblage information as well as clones available to the research community, a Web database with useful services was created at http://www.kazusa.or.jp/en/plant/chlamy/EST/.

Correlation of in vitro activity and in vivo efficacy of itraconazole intravenous and oral solubilized formulations by testing Candida strains with various itraconazole susceptibilities in a murine invasive infection

OBJECTIVES To examine whether in vitro antifungal susceptibility test results correlate with in vivo efficacy of two cyclodextrin-solubilized itraconazole formulations (intravenous and oral) against Candida in a murine model of invasive infection. METHODS A selected set of 12 Candida spp. strains with various itraconazole susceptibilities were tested. We studied the efficacy of intravenous and oral itraconazole administered once daily at dosages of 0.63, 2.5, 10 and 40 mg/kg body weight in mice lethally infected with each tested strain. Survival of mice in each treated group was monitored daily until the death of all control mice and compared between groups. RESULTS Survival of mice infected with 9 of 12 Candida strains with itraconazole MICs of ≤0.016-2.0 mg/L was significantly prolonged by treatment with intravenous itraconazole at dosages of 2.5 or 10 mg/kg and above. In contrast, the other three strains resistant to 8 mg/L itraconazole in vitro were refractory to the therapy, even at the highest itraconazole dosage (40 mg/kg). Closely similar in vivo data were obtained with the oral itraconazole therapy. The effective doses of the two itraconazole formulations increased with increasing itraconazole MICs for the infecting strains. CONCLUSIONS The in vivo efficacy of intravenous and oral itraconazole correlated with the in vitro susceptibility data.

Arsenate uptake by Pi transport system of an arsenate-resistant mutant, AR3, of Chlamydomonas

It is known that arsenate is taken up by the cells via Pi transport systems. In a green alga Chlamydomonas, arsenate was a competitive inhibitor of Pi transport systems, and the toxicity of arsenate disappeared in the presence of higher concentration of Pi. An arsenate-resistant mutant, AR3, which was generated by the random insertional mutagenesis, showed lower arsenic- and higher P-contents in the cells in the presence of arsenate than those of the wild type. The kinetics of Pi uptake demonstrated that AR3 had a high activity of a high-affinity Pi transport system even in the presence of 1mM Pi, where the system was suppressed in the wild type. The higher intracellular P content in AR3 might be due to one of the high-affinity Pi transporters. On the other hand, the kinetics of arsenate uptake indicated that the mutant had a high activity of a high-affinity arsenate transport system, which was suppressed rapidly after the incorporation in of arsenate into the cell. These results suggest that the arsenate resistance in AR3 is attributed to the high P content maintained by the activated Pi transport system and to the specific suppression of arsenate uptake via the Pi transport system.