Functional importance of Crenarchaea-specific extra-loop revealed by an X-ray structure of a heterotetrameric crenarchaeal splicing endonuclease

Archaeal splicing endonucleases (EndAs) are currently classified into three groups. Two groups require a single subunit protein to form a homodimer or homotetramer. The third group requires two nonidentical protein components for the activity. To elucidate the molecular architecture of the two-subunit EndA system, we studied a crenarchaeal splicing endonuclease from Pyrobaculum aerophilum. In the present study, we solved a crystal structure of the enzyme at 1.7-Å resolution. The enzyme adopts a heterotetrameric form composed of two catalytic and two structural subunits. By connecting the structural and the catalytic subunits of the heterotetrameric EndA, we could convert the enzyme to a homodimer that maintains the broad substrate specificity that is one of the characteristics of heterotetrameric EndA. Meanwhile, a deletion of six amino acids in a Crenarchaea-specific loop abolished the endonuclease activity even on a substrate with canonical BHB motif. These results indicate that the subunit architecture is not a major factor responsible for the difference of substrate specificity between single- and two-subunit EndA systems. Rather, the structural basis for the broad substrate specificity is built into the crenarchaeal splicing endonuclease itself

Effect of Inducers, Incubation Time and Heme Concentration on IC50 Value Variation in Anti-heme Crystallization Assay

Heme detoxification through crystallization into hemozoin has been suggested as a good target for the development of screening assays for new antimalarials. However, comparisons among the data obtained from different experiments are difficult, and the IC50 values (the concentrations of drug that are required to inhibit 50% of hemozoin formation) for the same drug vary widely. We studied the effects of changes in heme concentration (precursor of β-hematin), incubation time and three inducers (SDS, Tween 20 and linoleic acid) on the IC50 of some antimalarials (chloroquine, quinine, amodiaquine, and clotrimazole). The results showed that increasing both inducer concentration and incubation time raised the IC50 of selected antimalarials. Any change in those factors caused the IC50 value to vary. Standardization of assay conditions is, therefore, necessary to increase reproducibility and reduce discrepancies in assay performance. Considering all of the variables, the best choice of inducers is in the order of SDS > Tween 20 > linoleic acid

Selective cytotoxicity of dihydroorotate dehydrogenase inhibitors to human cancer cells under hypoxia and nutrient-deprived conditions

Human dihydroorotate dehydrogenase (HsDHODH) is a key enzyme of pyrimidine de novo biosynthesis pathway. It is located on the mitochondrial inner membrane and contributes to the respiratory chain by shuttling electrons to the ubiquinone pool. We have discovered ascofuranone (1), a natural compound produced by Acremonium sclerotigenum, and its derivatives are a potent class of HsDHODH inhibitors. We conducted a structure–activity relationship study and have identified functional groups of 1 that are essential for the inhibition of HsDHODH enzymatic activity. Furthermore, the binding mode of 1 and its derivatives to HsDHODH was demonstrated by co-crystallographic analysis and we show that these inhibitors bind at the ubiquinone binding site. In addition, the cytotoxicities of 1 and its potent derivatives 7, 8, and 9were studied using human cultured cancer cells. Interestingly, they showed selective and strong cytotoxicity to cancer cells cultured under microenvironment (hypoxia and nutrient-deprived) conditions. The selectivity ratio of 8 under this microenvironment show the most potent inhibition which was over 1000-fold higher compared to that under normal culture condition. Our studies suggest that under microenvironment conditions, cancer cells heavily depend on the pyrimidine de novo biosynthesis pathway. We also provide the first evidence that 1 and its derivatives are potential lead candidates for drug development which target the HsDHODH of cancer cells living under a tumor microenvironment

コウコウ シンカテイ オ ヘタ ガクセイ ニ タイスル ジョウホウ リテラシー キョウイク

高等学校において平成15年より施行された新課程では、高校のカリキュラムの中に「情報」科目が設けられた。これを経てきた学生に対する大学の情報リテラシー教育には、その位置づけや内容を再検討する必要が生じるかもしれない。そこで、高校新課程を経て大学に入学してきた学部1年生の学生を対象に、高校の情報教育で学んだ項目を調べることを目的としてアンケート調査を行った。大学の情報リテラシー科目において講義している項目を箇条書きにした用紙を配布して、高校の情報科目で学んだ項目に印をつけてもらい、その結果を集計した。本発表では、二つの大学で行ったアンケート調査の結果について述べ、これに基づいて考察した大学の情報リテラシー教育の対応について述べる。Due to the introduction of an information course to the curriculum of high school by the new Education Ministry guidelines, modifications might be required to information literacy education in universities. The terms learned in the information course of high school is investigated by questionnaires to the incoming freshmen of our universities. The present paper describes the result of an aggregate analysis of the questionnaires. The influences on the education of information literacy in universities caused by the introduction of the information course to high school are also discussed

ダイガクセイ ニ タイスル ジョウホウ リテラシー キョウイク

中学や高校で情報教育が行われるようになったことから、大学に入学してくる学生も既に情報教育やコンピューター利用経験を経てきている。大学の情報リテラシー教育もこれに対応する必要があると考え、新入学生に対して入学までに学んできた情報リテラシーを問うアンケート調査を、2006 年度より継続的に行っている。また、ワードプロセッシングについて、実技形式のアンケートも行っている。本稿では、二つの大学で行っているアンケート調査の結果について述べ、これに基づいて考察した大学の情報リテラシー教育について述べる。Due to the introduction of an information course to the curriculum of high school by the new Education Ministry guidelines,modifications may be required to information literacy education in universities. We have been investigated the terms learned in theinformation course of high school on the basis of a questionnaire to the incoming freshmen of our universities since 2006 scoolyear. Another questionnaire to test the skill of word presocessing is also examined. The present paper describes the results the twoquestionnaires, and discusses an appropriate curriculum of the infomation literacy education for university students

The ASCT/SCS cycle fuels mitochondrial ATP and acetate production in Trypanosoma brucei

Acetate:succinate CoA transferase (ASCT) is a mitochondrial enzyme that catalyzes the production of acetate and succinyl-CoA, which is coupled to ATP production with succinyl-CoA synthetase (SCS) in a process called the ASCT/SCS cycle. This cycle has been studied in Trypanosoma brucei (T. brucei), a pathogen of African sleeping sickness, and is involved in (i) ATP and (ii) acetate production and proceeds independent of oxygen and an electrochemical gradient. Interestingly, knockout of ASCT in procyclic form (PCF) of T. brucei cause oligomycin A-hypersensitivity phenotype indicating that ASCT/SCS cycle complements the deficiency of ATP synthase activity. In bloodstream form (BSF) of T. brucei, ATP synthase works in reverse to maintain the electrochemical gradient by hydrolyzing ATP. However, no information has been available on the source of ATP, although ASCT/ SCS cycle could be a potential candidate. Regarding mitochondrial acetate production, which is essential for fatty acid biosynthesis and growth of T. brucei, ASCT or acetyl-CoA hydrolase (ACH) are known to be its source. Despite the importance of this cycle, direct evidence of its function is lacking, and there are no comprehensive biochemical or structural biology studies reported so far. Here, we show that in vitro-reconstituted ASCT/SCS cycle is highly specific towards acetyl-CoA and has a higher k cat than that of yeast and bacterial ATP synthases. Our results provide the first biochemical basis for (i) rescue of ATP synthase-deficient phenotype by ASCT/SCS cycle in PCF and (ii) a potential source of ATP for the reverse reaction of ATP synthase in BSF.Voies métaboliques glycosomales non glycolytiques: nouvelles fonctions pour le développement et la virulence des trypanosomesAlliance française contre les maladies parasitaire

Structural and Biochemical Features of Eimeria tenella Dihydroorotate Dehydrogenase, a Potential Drug Target

Dihydroorotate dehydrogenase (DHODH) is a mitochondrial monotopic membrane protein that plays an essential role in the pyrimidine de novo biosynthesis and electron transport chain pathways. In Eimeria tenella, an intracellular apicomplexan parasite that causes the most severe form of chicken coccidiosis, the activity of pyrimidine salvage pathway at the intracellular stage is negligible and it relies on the pyrimidine de novo biosynthesis pathway. Therefore, the enzymes of the de novo pathway are considered potential drug target candidates for the design of compounds with activity against this parasite. Although, DHODHs from E. tenella (EtDHODH), Plasmodium falciparum (PfDHODH), and human (HsDHODH) show distinct sensitivities to classical DHODH inhibitors, in this paper,we identify ferulenol as a potent inhibitor of both EtDHODH and HsDHODH. Additionally, we report the crystal structures of EtDHODH and HsDHODH in the absence and presence of ferulenol. Comparison of these enzymes showed that despite similar overall structures, the EtDHODH has a long insertion in the N-terminal helix region that assumes a disordered configuration. In addition, the crystal structures revealed that the ferulenol binding pocket of EtDHODH is larger than that of HsDHODH. These differences can be explored to accelerate structure-based design of inhibitors specifically targeting EtDHODH

First Fabrication of GaInAs/InP Buried Metal Heterojunction Bipolar Transistor and Reduction of Base-Collector Capacitance

We report a novel approach for improving the performance of InP-based heterojunction bipolar transistors (HBTs). A buried-metal heterojunction bipolar transistor (BM-HBT), in which tungsten stripes of the same area as the emitter metal were buried in an i-InP collector layer, was fabricated for the first time. The aim in fabricating this structure is to realize a reduction in the total base-collector capacitance (C BCT ). In the measurement of microwave S-parameters, C BCT of 10.3 fF was evaluated. The effective base-collector junction area of the BM-HBT was estimated to be 22% that of conventional-HBT considering the difference in collector thickness