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Kiyoshi Kita

Publications and source records attributed to Kiyoshi Kita.

At least 19 recordsLinked to original sources
PubMed2007

Independent evolution of pyrimethamine resistance in Plasmodium falciparum isolates in Melanesia.

Pyrimethamine resistance in Plasmodium falciparum has previously been shown to have emerged once in Southeast Asia, from where it spread to Africa. Pyrimethamine resistance in this parasite is known to be conferred by mutations in the gene encoding dihydrofolate reductase (dhfr). We have analyzed polymorphisms in dhfr as well as microsatellite haplotypes flanking this gene in a total of 285 isolates from different regions of Melanesia (Papua New Guinea, Vanuatu, and the Solomon Islands) and Southeast Asia (Thailand and Cambodia). Nearly all isolates (92%) in Melanesia were shown to carry a dhfr double mutation (CNRNI [underlining indicates the mutation]) at positions 50, 51, 59, 108, and 164, whereas 98% of Southeast Asian isolates were either triple (CIRNI) or quadruple (CIRNL) mutants. Microsatellite analysis revealed two distinct lineages of dhfr double mutants in Melanesia. One lineage had the same microsatellite haplotype as that previously reported for Southeast Asia and Africa, suggesting the spread of this allele to Melanesia from Southeast Asia. The other lineage had a unique, previously undescribed microsatellite haplotype, indicative of the de novo emergence of pyrimethamine resistance in Melanesia.

Animals↗
PubMed2006

An evolutionary 'intermediate state' of mitochondrial translation systems found in Trichinella species of parasitic nematodes: co-evolution of tRNA and EF-Tu.

EF-Tu delivers aminoacyl-tRNAs to ribosomes in the translation system. However, unusual truncations found in some animal mitochondrial tRNAs seem to prevent recognition by a canonical EF-Tu. We showed previously that the chromadorean nematode has two distinct EF-Tus, one of which (EF-Tu1) binds only to T-armless aminoacyl-tRNAs and the other (EF-Tu2) binds to D-armless Ser-tRNAs. Neither of the EF-Tus can bind to canonical cloverleaf tRNAs. In this study, by analyzing the translation system of enoplean nematode Trichinella species, we address how EF-Tus and tRNAs have evolved from the canonical structures toward those of the chromadorean translation system. Trichinella mitochondria possess three types of tRNAs: cloverleaf tRNAs, which do not exist in chromadorean nematode mitochondria; T-armless tRNAs; and D-armless tRNAs. We found two mitochondrial EF-Tu species, EF-Tu1 and EF-Tu2, in Trichinella britovi. T.britovi EF-Tu2 could bind to only D-armless Ser-tRNA, as Caenorhabditis elegans EF-Tu2 does. In contrast to the case of C.elegans EF-Tu1, however, T.britovi EF-Tu1 bound to all three types of tRNA present in Trichinella mitochondria. These results suggest that Trichinella mitochondrial translation system, and particularly the tRNA-binding specificity of EF-Tu1, could be an intermediate state between the canonical system and the chromadorean nematode mitochondrial system.

Amino Acid Sequence↗
PubMed2006

Archaeal pre-mRNA splicing: a connection to hetero-oligomeric splicing endonuclease.

Eukaryotic Cbf5 is a protein subunit of the small nucleolar RNA-protein complex. Previously, we identified, in archaeal homologs of cbf5 of the crenarchaea, Aeropyrum pernix, Sulfolobus solfataricus, and Sulfolobus tokodaii, the first examples of introns of archaeal protein-coding genes. Here, we report the immunological detection of Cbf5 protein of S. tokodaii, the product of the spliced cbf5 mRNA. The hetero-oligomeric splicing endonuclease activity from recombinant S. tokodaii subunits cleaved at the exon-intron boundaries of cbf5 pre-mRNA fragments,suggesting that synthesis of full-length Cbf5 protein requires this activity. Database searches and PCR screens identified additional cbf5 introns in some, but not all sequenced crenarchaeal genomes. The predicted secondary structures of exon-intron boundaries of many of the newly identified intron-containing cbf5 pre-mRNAs contained relaxed forms of the bulge-helix-bulge motif similar to that of S. tokodaii. These observations are consistent with previous reports indicating that subunit composition of the splicing endonuclease contributes to substrate specificity.

Amino Acid Sequence↗
PubMed2006

Up-regulation of heme biosynthesis during differentiation of Neuro2a cells.

Heme is an iron-containing tetrapyrrole molecule that functions as a prosthetic group for proteins such as mitochondrial respiratory enzymes. Several studies have suggested that heme has essential functions in the construction and maintenance of the nervous system. In this study, the contents of three biologically important forms of heme (types a, b, and c) and the expression of heme biosynthetic enzymes were examined in differentiating Neuro2a cells. During neuronal differentiation, there were increases in the cellular heme levels and increases in the mRNA levels for the rate-limiting enzymes of heme biosynthesis, such as aminolevulinic acid synthase (ALAS; EC 2.3.1.37) and coproporphyrinogen oxidase (EC 1.3.3.3). With respect to heme contents, heme b increased in the late phase of differentiation, but no apparent increase in heme a or b was observed in the early phase. In contrast, heme c (cytochrome c) markedly increased during the early phase of differentiation. This change preceded the increase in heme b and the up-regulation of the mRNA levels for heme biosynthetic enzymes. This study suggests the up-regulation of heme biosynthesis and differential regulation of the heme a, b, and c levels during neuronal differentiation.

Animals↗
PubMed2006

Overexpression of peroxisome proliferator-activated receptor gamma co-activator-1alpha leads to muscle atrophy with depletion of ATP.

Peroxisome proliferator-activated receptor-gamma co-activator-1alpha (PGC-1alpha) is a key nuclear receptor co-activator for mitochondrial biogenesis. Here we report that overexpression of PGC-1alpha in skeletal muscles increased mitochondrial number and caused atrophy of skeletal muscle, especially type 2B fiber-rich muscles (gastrocnemius, quadriceps, and plantaris). Muscle atrophy became evident at 25 weeks of age, and a portion of the muscle was replaced by adipocytes. Mice showed increased energy expenditure and reduced body weight; thyroid hormone levels were normal. Mitochondria exhibited normal respiratory chain activity per mitochondrion; however, mitochondrial respiration was not inhibited by an ATP synthase inhibitor, oligomycin, clearly indicating that oxidative phosphorylation was uncoupled. Accordingly, ATP content in gastrocnemius was markedly reduced. A similar phenotype is observed in Luft's disease, a mitochondrial disorder that involves increased uncoupling of respiration and muscle atrophy. Our results indicate that overexpression of PGC-1alpha in skeletal muscle increases not only mitochondrial biogenesis but also uncoupling of respiration, resulting in muscle atrophy.

Adenosine Triphosphate↗
PubMed2005

PfPDE1, a novel cGMP-specific phosphodiesterase from the human malaria parasite Plasmodium falciparum.

This is the first report of molecular characterization of a novel cyclic nucleotide PDE (phosphodiesterase), isolated from the human malaria parasite Plasmodium falciparum and designated PfPDE1. PfPDE1 cDNA encodes an 884-amino-acid protein, including six putative transmembrane domains in the N-terminus followed by a catalytic domain. The PfPDE1 gene is a single-copy gene consisting of two exons and a 170 bp intron. PfPDE1 transcripts were abundant in the ring form of the asexual blood stages of the parasite. The C-terminal catalytic domain of PfPDE1, produced in Escherichia coli, specifically hydrolysed cGMP with a K(m) value of 0.65 microM. Among the PDE inhibitors tested, a PDE5 inhibitor, zaprinast, was the most effective, having an IC50 value of 3.8 microM. The non-specific PDE inhibitors IBMX (3-isobutyl-1-methylxanthine), theophylline and the antimalarial chloroquine had IC50 values of over 100 microM. Membrane fractions prepared from P. falciparum at mixed asexual blood stages showed potent cGMP hydrolytic activity compared with cytosolic fractions. This hydrolytic activity was sensitive to zaprinast with an IC50 value of 4.1 microM, but insensitive to IBMX and theophylline. Furthermore, an in vitro antimalarial activity assay demonstrated that zaprinast inhibited the growth of the asexual blood parasites, with an ED50 value of 35 microM. The impact of cyclic nucleotide signalling on the cellular development of this parasite has previously been discussed. Thus this enzyme is suggested to be a novel potential target for the treatment of the disease malaria.

Animals↗
PubMed2005

Chemotherapeutic efficacy of ascofuranone in Trypanosoma vivax-infected mice without glycerol.

Ascofuranone, an antibiotic isolated from Ascochyta visiae, showed trypanocidal activity in Trypanosoma vivax-infected mice. A single dose of 50 mg/kg ascofuranone effectively cured the mice without the help of glycerol. Repeated administrations of this drug further enhanced its chemotherapeutic effect. After two, three, and four consecutive days treatment, the doses needed to cure the infection decreased to 25, 12, and 6 mg/kg, so that the total doses administered were 50, 36 and 24 mg/kg, respectively. Ascofuranone (50 mg/kg) also had a prophylactic effect against T. vivax infection within the first two days after administration. This prophylactic activity diminished to 80% by day 3 and completely disappeared four days after administration. Of particular interest in this study was that ascofuranone had trypanocidal activity in T. vivax-infected mice in the absence of glycerol, whereas co-administration of glycerol or repeated administrations of this drug are needed for Trypanosoma brucei brucei infection. Our present results strongly suggest that ascofuranone is also an effective tool in chemotherapy against African trypanosomiasis in domestic animals.

Animals↗
PubMed2005

Kinetics and strain variation of phagosome proteins of Entamoeba histolytica by proteomic analysis.

The protozoan parasite Entamoeba histolytica ingests and feeds on microorganisms and mammalian cells. Phagocytosis is essential for cell growth and implicated in pathogenesis of E. histolytica. We report here the dynamic changes of phagosome proteins during phagosome maturation by proteomic analysis using reversed-phase capillary liquid chromatography and ion trap tandem mass spectrometry. Phagosomes were isolated at various intervals after internalization of latex beads. Immunoblot analysis and electron microscopy verified successful isolation of phagosomes. A total of 159 proteins were identified from the reference strain HM1 at different stages of phagosome maturation. Approximately 70% of them were detected in a time-dependent fashion, suggesting dynamism of phagosome biogenesis. The kinetics of representative proteins were verified by immunoblots and also by video microscopy of live transgenic amebae expressing green fluorescent protein-fused EhRab7A. Furthermore, we observed significant differences in phagosome profiles between HM1 and two recent clinical isolates. Approximately 60% of 229 proteins detected in at least one of these three strains were identified only in one strain, while approximately 20% of these proteins were detected in all three strains. These data should provide significant insights into molecular characterization of phagosome biogenesis, and help to elucidate the pathogenesis of this important infection.

Animals↗
PubMed2005

Identification and characterization of amino acid residues essential for the active site of UDP-N-acetylenolpyruvylglucosamine reductase (MurB) from Staphylococcus aureus.

The enzymes essential for bacterial peptidoglycan biosynthesis are attractive targets for antimicrobial drug development. One of these is MurB, which contains FAD as a cofactor and catalyzes the NADPH-dependent reduction of UDP-N-acetylenolpyruvylglucosamine (UDP-GlcNAcEP) to UDP-N-acetylmuramic acid. This study examined the roles of the conserved amino acid residues of Staphylococcus aureus MurB, which are located near the active site in x-ray crystal structures. Seven of 11 site-directed mutated murB genes lost the ability to complement a temperature-sensitive S. aureus murB mutant. Biochemical characterization of the seven mutated MurB proteins revealed that they cannot carry out the reduction of UDP-GlcNAcEP, although they can all catalyze the intramolecular reduction of FAD via NADPH. Spectrometric analyses of the oxidized form of the mutated proteins in the presence and absence of NADP+ or UDP-GlcNAcEP revealed that these essential amino acid residues play four distinct roles in substrate interactions: Arg213 is essential for maintenance of the electronic state of FAD; Arg176 is required for interaction with UDP-GlcNAcEP; His259 is required for interaction with both UDP-GlcNAcEP and NADP+; and Asn71, Tyr175, Ser226, and Glu296 are not apparently required for interaction with either ligand. The results presented here identify for the first time the amino acid residues of MurB that are required for the interaction with UDP-Glc-NAcEP and NADP+.

Amino Acid Sequence↗
PubMed2005

Genetic diversity and kinetic properties of Trypanosoma cruzi dihydroorotate dehydrogenase isoforms.

Dihydroorotate dehydrogenase (DHOD) is the fourth enzyme in the de novo pyrimidine biosynthetic pathway and is essential in Trypanosoma cruzi, the parasitic protist causing Chagas' disease. T. cruzi and human DHOD have different biochemical properties, including the electron acceptor capacities and cellular localization, suggesting that T. cruzi DHOD may be a potential chemotherapeutic target against Chagas' disease. Here, we report nucleotide sequence polymorphisms of T. cruzi DHOD genes and the kinetic properties of the recombinant enzymes. T. cruzi Tulahuen strain possesses three DHODgenes: DHOD1 and DHOD2, involved in the pyrimidine biosynthetic (pyr) gene cluster on an 800 and a 1000 kb chromosomal DNA, respectively, and DHOD3, located on an 800 kb DNA. The open reading frames of all three DHOD genes are comprised of 942 bp, and encode proteins of 314 amino acids. The three DHOD genes differ by 26 nucleotides, resulting in replacement of 8 amino acid residues. In contrast, all residues critical for constituting the active site are conserved among the three proteins. Recombinant T. cruzi DHOD1 and DHOD2 expressed in E. coli possess similar enzymatic properties, including optimal pH, optimal temperature, Vmax, and Km for dihydroorotate and fumarate. In contrast, DHOD3 had a higher Vmax and Km for both substrates. Orotate competitively inhibited all three DHOD enzymes to a comparable level. These results suggest that, despite their genetic variations, kinetic properties of the three T. cruziDHODs are conserved. Our findings facilitate further exploitation of T. cruzi DHOD inhibitors, as chemotherapeutic agents against Chagas' disease.

Animals↗
PubMed2005

Expression, purification and crystallization of Trypanosoma cruzi dihydroorotate dehydrogenase complexed with orotate.

Dihydroorotate dehydrogenase (DHOD) catalyzes the oxidation of dihydroorotate to orotate, the fourth step and the only redox reaction in the de novo biosynthesis of pyrimidine. DHOD from Trypanosoma cruzi (TcDHOD) has been expressed as a recombinant protein in Escherichia coli and purified to homogeneity. Crystals of the TcDHOD-orotate complex were grown at 277 K by the sitting-drop vapour-diffusion technique using polyethylene glycol 3350 as a precipitant. The crystals diffract to better than 1.8 A resolution using synchrotron radiation (lambda = 0.900 A). X-ray diffraction data were collected at 100 K and processed to 1.9 A resolution with 98.2% completeness and an overall Rmerge of 7.8%. The TcDHOD crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 67.87, b = 71.89, c = 123.27 A. The presence of two molecules in the asymmetric unit (2 x 34 kDa) gives a crystal volume per protein weight (VM) of 2.2 A3 Da(-1) and a solvent content of 44%.

Aminomethyltransferase↗
PubMed2005

Functional reconstitution of a crenarchaeal splicing endonuclease in vitro.

Sulfolobus tokodaii strain 7 is one of Crenarchaea whose entire genome has been sequenced. The genome sequence revealed that it possesses two open reading frames (ORFs) that are homologous to EndA, a protein responsible for splicing endonuclease activity in Archaea. Interestingly, one of the two ORFs lacks a putative catalytic amino acid residue for the nuclease activity. To investigate their functions, the two ORF products were individually expressed in Escherichia coli, partially purified, and tested for their nuclease activities in vitro. Using in vitro transcribed tRNA precursor as a substrate, we found that the two ORF products are concurrently required to cleave exon-intron junctions. Our finding implies that the splicing endonuclease for the organism is a multi-subunit complex composed of the two endA gene products.

Amino Acid Sequence↗
PubMed2005

Mutational analysis of the Trypanosoma vivax alternative oxidase: the E(X)6Y motif is conserved in both mitochondrial alternative oxidase and plastid terminal oxidase and is indispensable for enzyme activity.

Based on amino acid sequence similarity and the ability to catalyze the four-electron reduction of oxygen to water using a quinol substrate, mitochondrial alternative oxidase (AOX) and plastid terminal oxidase (PTOX) appear to be two closely related members of the membrane-bound diiron carboxylate group of proteins. In the current studies, we took advantage of the high activity of Trypanosoma vivax AOX (TvAOX) to examine the importance of the conserved Glu and the Tyr residues around the predicted third helix region of AOXs and PTOXs. We first compared the amino acid sequences of TvAOX with AOXs and PTOXs from various taxa and then performed alanine-scanning mutagenesis of TvAOX between amino acids Y(199) and Y(247). We found that the ubiquinol oxidase activity of TvAOX is completely lost in the E214A mutant, whereas mutants E215A and E216A retained more than 30% of the wild-type activity. Among the Tyr mutants, a complete loss of activity was also observed for the Y221A mutant, whereas the activities were equivalent to wild-type for the Y199A, Y212A, and Y247A mutants. Finally, residues Glu(214) and Tyr(221) were found to be strictly conserved among AOXs and PTOXs. Based on these findings, it appears that AOXs and PTOXs are a novel subclass of diiron carboxylate proteins that require the conserved motif E(X)(6)Y for enzyme activity.

Amino Acid Motifs↗
PubMed2005

Isolation and physiochemical properties of protein-rich nematode mitochondrial ribosomes.

In the present study, mitochondrial ribosomes of the nematode Ascaris suum were isolated and their physiochemical properties were compared to ribosomes of Escherichia coli. The sedimentation coefficient and buoyant density of A. suum mitochondrial ribosomes were determined. The sedimentation coefficient of the intact monosome was about 55 S. The buoyant density of formaldehyde-fixed ribosomes in cesium chloride was 1.40 g/cm(3), which suggests that the nematode mitoribosomes have a much higher protein composition than other mitoribosomes. The diffusion coefficients obtained from dynamic light scattering measurements were (1.48 +/- 0.04) x 10(-)(7) cm(2) s(-)(1) for 55 S mitoribosomes and (1.74 +/- 0.04) x 10(-)(7) cm(2) s(-)(1) for the 70 S E. coli monosome. The diameter of mitoribosomes was measured by dynamic light-scattering analysis and electron microscopy. Though the nematode mitoribosome has a larger size than the bacterial ribosome, it does not differ significantly in size from mammalian mitoribosomes.

Animals↗
PubMed2005

Expression of mRNAs and proteins for peroxiredoxins in Plasmodium falciparum erythrocytic stage.

mRNA and protein expression profiles for three peroxiredoxins (PfTPx-1, PfTPx-2 and Pf1-Cys-Prx) and a thioredoxin (PfTrx-1) of Plasmodium falciparum during the erythrocytic stage were examined by real-time quantitative reverse transcription-PCR (RT-PCR), Western blotting and confocal laser scanning microscopy. PfTPx-1 was expressed constitutively in the parasite cytoplasm throughout the erythrocytic stage, suggesting a housekeeping role of this enzyme for control of intercellular reactive oxygen species (ROS) in the parasite. Pf1-Cys-Prx showed elevated expression during the trophozoite and early schizont stages in the parasite cytoplasm, and this profile suggested that this peroxiredoxin (Prx) detoxifies metabolism-derived ROS such as those released from heme iron. The other 2-Cys Prx, PfTPx-2, was detected in mitochondria and was expressed in both the trophozoite and schizont stages. Detection of the Prx in mitochondria is consistent with recent reports of the existence of a respiratory chain, which produces ROS, in the mitochondria of P. falciparum. PfTrx-1 showed elevated expression during the trophozoite and schizont stages in the parasite cytoplasm. Finally, expression of these antioxidant protein genes is most likely regulated at the transcriptional level because their mRNA and protein expression profiles overlapped.

Animals↗
PubMed2005

A gamma-lactone form nafuredin, nafuredin-gamma, also inhibits helminth complex I.

Nafuredin, a delta-lactone antibiotic, is a fungal metabolite showing selective helminth NADH-fumarate reductase inhibition, and whose target had been revealed as complex I. We found that nafuredin is easily converted to nafuredin-gamma by weak alkaline treatment. The structure of nafuredin-gamma was elucidated as a gamma-lactone form of nafuredin with keto-enol tautomerism. Nafuredin-gamma shows similar complex I inhibitory activity as nafuredin, and it also possesses anthelmintic activity in vivo.

Animals↗
PubMed2005

Functional reconstitution of an archaeal splicing endonuclease in vitro.

Sulfolobus tokodaii strain 7 is one of archaea whose entire genome has been sequenced. The genome sequence revealed that it possesses two open reading frames (ORFs) that are homologous to endA, a protein responsible for splicing endonuclease activity in archaea. Interestingly, one of these two ORFs lacks a putative catalytic amino acid residue for the nuclease activity. To investigate their functions, the two ORFs were individually expressed in E. coli, partially purified, and tested for their nuclease activities in vitro. Using in vitro transcribed tRNA precursors as substrates, we found that the two ORF products are concurrently required to cleave exon-intron junctions. Our finding implies that the splicing endonuclease of the organism is a multi-subunit complex composed of the two ORF products.

Archaeal Proteins↗