Publications by authors named "Akio Ueno"

36 Publications

sp. nov., a mesophilic sulfate-reducing deltaproteobacterium isolated from a deep siliceous mudstone formation.

Int J Syst Evol Microbiol 2021 Feb;71(2)

Horonobe Research Institute for the Subsurface Environment (H-RISE) Northern Advancement Centre for Science and Technology (NOASTEC), Sakae-machi, Horonobe-cho, Teshio-gun, Hokkaido, Japan.

A novel mesophilic sulfate-reducing bacterium, strain HN2, was isolated from groundwater sampled from the subsurface siliceous mudstone of the Wakkanai Formation located in Horonobe, Hokkaido, Japan. The bacterium was Gram-negative and vibrio-shaped, and its motility was conferred by a single polar flagellum. Cells had desulfoviridin. Catalase and oxidase activities were not detected. It grew in the temperature range of 25-40 °C (optimum, 35 °C) and pH range of 6.3-8.1 (optimum, pH 7.2-7.6). It used sulfate, thiosulfate, dimethyl sulfoxide, anthraquinone-2,6-disulfonate, Fe, and manganese oxide, but not elemental sulfur, nitrite, nitrate, or fumarate as electron acceptors. The strain showed weak growth with sulfite as the electron acceptor. Fermentative growth with pyruvate, lactate and cysteine was observed in the absence of sulfate, but not with malate or fumarate. NaCl was not required, but the strain tolerated up to 40 g l. Strain HN2 did not require vitamins. The major cellular fatty acids were iso-C (23.8 %), C9 (18.4 %), C (15.0 %), C (14.5 %), and anteiso-C (10.1 %). The major respiratory quinone was menaquinone MK-6(H). The G+C content of the genomic DNA was 56.7 mol%. Based on 16S rRNA gene sequence analysis, the closest phylogenetic relative of strain HN2 is JS1 (97.0 %). Digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values of the strains HN2 and JS1 were 22.2 and 79.8 %, respectively. Based on the phenotypic and molecular genetic evidence, we propose a novel species, sp. nov. with the type strain HN2 (=DSM 101010=NBRC 112213).
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http://dx.doi.org/10.1099/ijsem.0.004683DOI Listing
February 2021

Evaluation of novel oocyst wall protein candidates of Toxoplasma gondii.

Parasitol Int 2017 Oct 30;66(5):643-651. Epub 2017 May 30.

National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Hokkaido 080-8555, Japan. Electronic address:

Oocyst stage of Toxoplasma gondii is characterized by a durable wall that confers a strong protection to this protozoan parasite in face of harsh environmental conditions. Thus, it is considered the key for transmission of T. gondii. Analysis of oocyst wall composition is mandatory therefore; the aim of this study was to identify novel T. gondii oocyst wall proteins and test their use in detection of these oocysts in environmental samples. Five candidates of novel T. gondii oocyst wall proteins (TgOWPs) were identified and named TgOWP8 through TgOWP12. Recombinant protein of TgOWP8 was expressed in E. coli using glutathione S-transferase as fusion protein. Polyclonal antibody was produced and validated by indirect immunofluorescence antibody assay (IFA). For detection by IFA, we used different methods for fixation and permeabilization of oocysts to improve the antigen-antibody detection. Specificity to wall of T. gondii oocyst was confirmed and revealed absence of cross reactivity with bradyzoite cyst wall and tachyzoites. Although some TgOWPs were identified previously, our study represents a continuation of molecular investigations of oocyst wall proteins as an essential structure for the longevity and infectivity of this stage and also provided new trial to improve T. gondii oocysts detection.
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http://dx.doi.org/10.1016/j.parint.2017.05.009DOI Listing
October 2017

Effects of Aerobic Growth on the Fatty Acid and Hydrocarbon Compositions of Geobacter bemidjiensis Bem.

J Oleo Sci 2017 Jan 8;66(1):93-101. Epub 2016 Dec 8.

Horonobe Research Institute for the Subsurface Environment (H-RISE), NOASTEC.

Geobacter spp., regarded as strict anaerobes, have been reported to grow under aerobic conditions. To elucidate the role of fatty acids in aerobiosis of Geobacter spp., we studied the effect of aerobiosis on fatty acid composition and turnover in G. bemidjiensis Bem. G. bemidjiensis Bem was grown under the following different culture conditions: anaerobic culture for 4 days (type 1) and type 1 culture followed by 2-day anaerobic (type 2) or aerobic culture (anaerobic-to-aerobic shift; type 3). The mean cell weight of the type 3 culture was approximately 2.5-fold greater than that of type 1 and 2 cultures. The fatty acid methyl ester and hydrocarbon fraction contained hexadecanoic (16:0), 9-cis-hexadecenoic [16:1(9c)], tetradecanoic (14:0), tetradecenoic [14:1(7c)] acids, hentriacontanonaene, and hopanoids, but not long-chain polyunsaturated fatty acids. The type 3 culture contained higher levels of 14:0 and 14:1(7c) and lower levels of 16:0 and 16:1(9c) compared with type 1 and 2 cultures. The weight ratio of extracted lipid per dry cell was lower in the type 3 culture than in the type 1 and 2 cultures. We concluded that anaerobically-grown G. bemidjiensis Bem followed by aerobiosis were enhanced in growth, fatty acid turnover, and de novo fatty acid synthesis.
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http://dx.doi.org/10.5650/jos.ess16122DOI Listing
January 2017

Bacterial Long-Chain Polyunsaturated Fatty Acids: Their Biosynthetic Genes, Functions, and Practical Use.

Mar Drugs 2016 May 12;14(5). Epub 2016 May 12.

Laboratory of Environmental Microbiology, Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Kita-ku, Sapporo, Hokkaido 060-8589, Japan.

The nutritional and pharmaceutical values of long-chain polyunsaturated fatty acids (LC-PUFAs) such as arachidonic, eicosapentaenoic and docosahexaenoic acids have been well recognized. These LC-PUFAs are physiologically important compounds in bacteria and eukaryotes. Although little is known about the biosynthetic mechanisms and functions of LC-PUFAs in bacteria compared to those in higher organisms, a combination of genetic, bioinformatic, and molecular biological approaches to LC-PUFA-producing bacteria and some eukaryotes have revealed the notably diverse organization of the pfa genes encoding a polyunsaturated fatty acid synthase complex (PUFA synthase), the LC-PUFA biosynthetic processes, and tertiary structures of the domains of this enzyme. In bacteria, LC-PUFAs appear to take part in specific functions facilitating individual membrane proteins rather than in the adjustment of the physical fluidity of the whole cell membrane. Very long chain polyunsaturated hydrocarbons (LC-HCs) such as hentriacontanonaene are considered to be closely related to LC-PUFAs in their biosynthesis and function. The possible role of LC-HCs in strictly anaerobic bacteria under aerobic and anaerobic environments and the evolutionary relationships of anaerobic and aerobic bacteria carrying pfa-like genes are also discussed.
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http://dx.doi.org/10.3390/md14050094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4882568PMC
May 2016

Identification and characterization of profilin antigen among Babesia species as a common vaccine candidate against babesiosis.

Exp Parasitol 2016 Jul 19;166:29-36. Epub 2016 Mar 19.

National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido, 080-8555, Japan. Electronic address:

We have characterized a member of the profilin (PROF) family protein as a common antigen in three pathogens-Babesia bovis (B. bovis), Babesia bigemina (B. bigemina), and Babesia microti (B. microti)-and evaluated its immunogenic and cross-protective properties against a challenge infection with B. microti in BALB/c mice. The recombinant PROF proteins of B. bovis, B. bigemina, and B. microti were successfully expressed in Escherichia coli (E. coli) as soluble GST fusion proteins (rBboPROF, rBbigPROF, and rBmPROF, respectively), and they were found to be antigenic. On probing with mouse anti-rPROF serum, green fluorescence was observed on the parasites' cytosols by confocal laser microscopy. Immunization regimes in BALB/c mice using rPROFs induced cross-protective immunity against B. microti infection based on high levels of cytokines and immunoglobulin (IgG) titers, a reduction in peak parasitemia levels, and earlier clearance of the parasite as compared with control mice. The findings of the present study indicate that PROF is a common antigen among bovine and murine Babesia parasites, and it might be used as a common vaccine candidate against babesiosis.
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http://dx.doi.org/10.1016/j.exppara.2016.03.024DOI Listing
July 2016

Anaerobic decomposition of humic substances by Clostridium from the deep subsurface.

Sci Rep 2016 Jan 8;6:18990. Epub 2016 Jan 8.

Horonobe Research Institute for the Subsurface Environment, Northern Advancement Centre for Science and Technology, 5-3, Sakae-machi, Horonobe-cho, Teshio-gun, Hokkaido 098-3221, Japan.

Decomposition of humic substances (HSs) is a slow and cryptic but non-negligible component of carbon cycling in sediments. Aerobic decomposition of HSs by microorganisms in the surface environment has been well documented; however, the mechanism of anaerobic microbial decomposition of HSs is not completely understood. Moreover, no microorganisms capable of anaerobic decomposition of HSs have been isolated. Here, we report the anaerobic decomposition of humic acids (HAs) by the anaerobic bacterium Clostridium sp. HSAI-1 isolated from the deep terrestrial subsurface. The use of (14)C-labelled polycatechol as an HA analogue demonstrated that the bacterium decomposed this substance up to 7.4% over 14 days. The decomposition of commercial and natural HAs by the bacterium yielded lower molecular mass fractions, as determined using high-performance size-exclusion chromatography. Fourier transform infrared spectroscopy revealed the removal of carboxyl groups and polysaccharide-related substances, as well as the generation of aliphatic components, amide and aromatic groups. Therefore, our results suggest that Clostridium sp. HSAI-1 anaerobically decomposes and transforms HSs. This study improves our understanding of the anaerobic decomposition of HSs in the hidden carbon cycling in the Earth's subsurface.
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http://dx.doi.org/10.1038/srep18990DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4705541PMC
January 2016

Characterization of Toxoplasma gondii glyoxalase 1 and evaluation of inhibitory effects of curcumin on the enzyme and parasite cultures.

Parasit Vectors 2015 Dec 23;8:654. Epub 2015 Dec 23.

National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, 080-8555, Japan.

Background: The glyoxalase pathway, which includes two enzymes, glyoxalase 1 and 2 (Glo1 and Glo2), is a ubiquitous cellular system responsible for the removal of cytotoxic methylglyoxal produced during glycolysis. Protozoan parasites, including Toxoplasma gondii (T. gondii) tachyzoites, produce methylglyoxal because of increased glycolytic fluxes. A Glo1 inhibitor such as curcumin could be considered a drug candidate for anti-protozoan, anti-inflammatory, and anti-cancer therapy.

Methods: The T. gondii Glo1 gene (TgGlo1) was cloned and the recombinant protein was produced. Enzyme kinetics of TgGlo1 and five mutants were evaluated by adding methylglyoxal and glutathione to a reaction mixture. Finally, the inhibitory effects of various concentrations of curcumin on recombinant TgGlo1 were evaluated using in vitro cultures of T. gondii.

Results: Active recombinant TgGlo1 was successfully produced and the active sites (E166 and E251) of TgGlo1 were verified by point mutagenesis. Curcumin at the tested doses inhibited the enzymatic activity of recombinant TgGlo1 as well as the parasitic propagation of in vitro-cultured T. gondii. The Ki and IC50 were 12.9 ± 0.5 μM and 38.3 ± 0.9 μM, respectively.

Conclusion: The inhibitory effect of curcumin on the enzymatic activity of TgGlo1 and parasitic propagation of T. gondii could be explored in the potential development of a potent drug for the treatment of toxoplasmosis. However, considering the fact that curcumin is known to have many effects on other molecules in the micromolar range, further elucidation of curcumin's direct inhibition of the glyoxalase system of T. gondii will be needed.
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http://dx.doi.org/10.1186/s13071-015-1268-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4688987PMC
December 2015

Methanosarcina subterranea sp. nov., a methanogenic archaeon isolated from a deep subsurface diatomaceous shale formation.

Int J Syst Evol Microbiol 2015 Apr 20;65(Pt 4):1167-1171. Epub 2015 Jan 20.

Horonobe Research Institute for the Subsurface Environment, Northern Advancement Center for Science and Technology, Horonobe-cho, Teshio-gun, Hokkaido 098-3221, Japan.

A methanogenic archaeon, strain HC-2(T), was isolated from a deep diatomaceous shale formation. The strain grew on methanol, monomethylamine, dimethylamine, trimethylamine and dimethylsulphide, but not on acetate, H2/CO2, formate, 2-propanol, 2-butanol or cyclopentanol. Cells were Gram-stain-negative, non-motile, and coccus-like, 0.9-1.4 µm in diameter, and occurred singly, in pairs, or as aggregates. The strain grew at 10-40 °C (optimum 35 °C), pH 5.9-7.4 (optimum pH 6.6-6.8) and in 0-0.6 M NaCl (optimum 0.1-0.2 M). The genomic DNA G+C content was 41.5 mol% and the 16S rRNA gene sequence was closely related to those of Methanosarcina lacustris DSM 13486(T) (99.1%) and Methanosarcina siciliae DSM 3028(T) (98.3%). Values for DNA-DNA hybridization with these strains were less than 30%. The phenotypic and phylogenetic features of HC-2(T) indicate that it represents a novel species of the genus Methanosarcina , for which the name Methanosarcina subterranea sp. nov. is proposed. The type strain is HC-2(T) ( = DSM 22503(T) = JCM 15540(T) = NBRC 102578(T)).
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http://dx.doi.org/10.1099/ijs.0.000072DOI Listing
April 2015

Molecular characterization of a new Babesia bovis thrombospondin-related anonymous protein (BbTRAP2).

PLoS One 2013 13;8(12):e83305. Epub 2013 Dec 13.

National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, Japan.

A gene encoding a Babesia bovis protein that shares significant degree of similarity to other apicomplexan thrombospondin-related anonymous proteins (TRAPs) was found in the genomic database and designated as BbTRAP2. Recombinant protein containing a conserved region of BbTRAP2 was produced in E. coli. A high antigenicity of recombinant BbTRAP2 (rBbTRAP2) was observed with field B. bovis-infected bovine sera collected from geographically different regions of the world. Moreover, antiserum against rBbTRAP2 specifically reacted with the authentic protein by Western blot analysis and an indirect fluorescent antibody test. Three bands corresponding to 104-, 76-, and 44-kDa proteins were identified in the parasite lysates and two bands of 76- and 44-kDa proteins were detected in the supernatant of cultivated parasites, indicating that BbTRAP2 was proteolytically processed and shed into the culture. Apical and surface localizations of BbTRAP2 were observed in the intracellular and extracellular parasites, respectively, by confocal laser microscopic examination. Moreover, native BbTRAP2 was precipitated by bovine erythrocytes, suggesting its role in the attachment to erythrocytes. Furthermore, the specific antibody to rBbTRAP2 inhibited the growth of B. bovis in a concentration-dependent manner. Consistently, pre-incubation of the free merozoites with the antibody to rBbTRAP2 resulted in an inhibition of the parasite invasion into host erythrocytes. Interestingly, the antibody to rBbTRAP2 was the most inhibitive for the parasite's growth as compared to those of a set of antisera produced against different recombinant proteins, including merozoite surface antigen 2c (BbMSA-2c), rhoptry-associated protein 1 C-terminal (BbRAP-1CT), and spherical body protein 1 (BbSBP-1). These results suggest that BbTRAP2 might be a potential candidate for development of a subunit vaccine against B. bovis infection.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0083305PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3862764PMC
September 2014

Methanoculleus horonobensis sp. nov., a methanogenic archaeon isolated from a deep diatomaceous shale formation.

Int J Syst Evol Microbiol 2013 Nov 5;63(Pt 11):4320-4323. Epub 2013 Jul 5.

Horonobe Research Institute for the Subsurface Environment, Northern Advancement Center for Science and Technology, Horonobe-cho, Teshio-gun, Hokkaido 098-3221, Japan.

A methanogenic organism from the domain Archaea, designated strain T10(T), was isolated from groundwater sampled from a deep diatomaceous shale formation located in Horonobe, Hokkaido, Japan. The strain utilized H2/CO2 and formate as substrates for methanogenesis. Cells were strictly anaerobic, Gram-negative-staining, flagellated, irregular coccoids, 0.7-1.6 µm in diameter, and occurred singly. The strain grew at 25-45 °C (optimum 37-42 °C), at pH 5.8-8.2 (optimum pH 6.7-6.8) and in the presence of 0-1.3 M NaCl (optimum 0.1-0.2 M NaCl). The G+C content of the genomic DNA was 62.9 mol%. 16S rRNA gene sequencing revealed that, although the strain is a member of the genus Methanoculleus, it clearly differed from all described species of this genus (95.5-98.3 % sequence similarity). Values for DNA-DNA hybridization with type strains of closely related Methanoculleus species were less than 50 %. Phenotypic and phylogenetic features of strain T10(T) clearly indicate that it represents a novel species of the genus Methanoculleus, for which the name Methanoculleus horonobensis sp. nov. is proposed. The type strain is T10(T) ( = DSM 21626(T) = JCM 15517(T)).
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http://dx.doi.org/10.1099/ijs.0.053520-0DOI Listing
November 2013

Actin polymerization mediated by Babesia gibsoni aldolase is required for parasite invasion.

Exp Parasitol 2013 Sep 17;135(1):42-9. Epub 2013 Jun 17.

National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan.

Host cell invasion by apicomplexan parasites driven by gliding motility and empowered by actin-based movement is essential for parasite survival and pathogenicity. The parasites share a conserved invasion process: actin-based motility led by the coordination of adhesin-cytoskeleton via aldolase. A number of studies of host cell invasion in the Plasmodium species and Toxoplasma gondii have been performed. However, the mechanisms of host cell invasion by Babesia species have not yet been studied. Here, we show that Babesia gibsoni aldolase (BgALD) forms a complex with B. gibsoni thrombospondin-related anonymous protein (BgTRAP) and B. gibsoni actin (BgACT), depending on tryptophan-734 (W-734) in BgTRAP. In addition, actin polymerization is mediated by BgALD. Moreover, cytochalasin D, which disrupts actin polymerization, suppressed B. gibsoni parasite growth and inhibited the host cell invasion by parasites, indicating that actin dynamics are essential for erythrocyte invasion by B. gibsoni. This study is the first molecular approach to determine the invasion mechanisms of Babesia species.
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http://dx.doi.org/10.1016/j.exppara.2013.06.002DOI Listing
September 2013

A novel dense granule protein, GRA22, is involved in regulating parasite egress in Toxoplasma gondii.

Mol Biochem Parasitol 2013 May 23;189(1-2):5-13. Epub 2013 Apr 23.

National Research Center for Protozoan Diseases (NRCPD), Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan.

The intracellular protozoan parasite Toxoplasma gondii is capable of invading any nucleated cell and replicates within a parasitophorous vacuole (PV). This microenvironment is modified by secretory proteins from organelles named rhoptries and dense granules. In this report, we identify a novel dense granule protein, which we refer to as GRA22. GRA22 has no significant homology to any other known proteins. GRA22 possesses a signal peptide at the N-terminal end which is responsible for dense granule and PV localization. The RH strain GRA22 contains 12 copies of tandem repeats consisting each of 21 amino acids located between the 42nd and 293rd amino acid residues from a full length of 624 amino acids. On the other hand, ME49 strain GRA22 has 10 copies of tandem repeats. The Neospora caninum GRA22 ortholog completely lacks this repetitive sequence. GRA22 knock out parasites show a similar growth rate as the parental strain. However, the timing of egress is earlier than that of the parental strain. These results suggest that GRA22 is involved in regulating parasite egress in T. gondii.
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http://dx.doi.org/10.1016/j.molbiopara.2013.04.005DOI Listing
May 2013

Farsenyl pyrophosphate synthase is a potential molecular drug target of risedronate in Babesia bovis.

Parasitol Int 2013 Apr 28;62(2):189-92. Epub 2012 Dec 28.

National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, 2-13 Inada-cho, Obihiro, Hokkaido, Japan.

A cDNA encoding farnesyl pyrophosphate synthase of Babesia bovis (BbFPPS) has been isolated, cloned and characterized as molecular drug target. Sequence analysis revealed that BbFPPS contains an open reading frame of 1011bp with predicted 336 amino acids and molecular mass of 38kDa. Antiserum raised in mice against recombinant BbFPPS expressed in Escherichia coli specifically reacted with native protein of B. bovis parasites by Western blot analysis and indirect immunofluorescent test. Enzymatic assay using recombinant BbFPPS revealed that the Km value of the enzyme for isopentenyl pyrophosphate and dimethylallyl pyrophosphate was 2.494±1.536μM. Risedronate inhibited the activity of BbFPPS yielding IC50 value of 8.4±1.2nM. Furthermore, the in vitro growth of B. bovis was significantly inhibited in the presence of a micromolar concentration of risedronate (IC50=4.02±0.91μM). No regrowth of B. bovis was observed at 10μM of risedronate in the subsequent viability test. These results demonstrate that BbFPPS is the molecular target of risedronate, which could inhibit the in vitro growth of B. bovis.
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http://dx.doi.org/10.1016/j.parint.2012.12.005DOI Listing
April 2013

Cloning and characterization of histone deacetylase from Babesia bovis.

Vet Parasitol 2012 Dec 29;190(3-4):423-33. Epub 2012 Jun 29.

National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan.

The effect of inhibitors of histone deacetylase (HDAC) on Apicomplexa has been previously reported with the discovery of apicidin, a cyclic tetrapeptide having broad-spectrum antiparasitic activity. In the current study, we expressed Babesia bovis (B. bovis) recombinant-HDAC 3 (rBbHDAC3) as a GST-fusion protein in Escherichia coli (E. coli) and found that it was antigenic. An antiserum against the recombinant protein was generated in mice. The mice serum demonstrated the presence of HDAC in B. bovis by a Western blot assay. The murine anti-rBbHDAC3 reacted with B. bovis, Babesia bigemina (B. bigemina), Theileria equi (T. equi), and Babeisa caballi (B. caballi) merozoites in the indirect fluorescent antibody test (IFAT). Furthermore, the HDAC-enzymatic activity of the rBbHDAC3 protein was evaluated by a colorimetric assay. The enzymatic activity of rBbHDAC3 was inhibited by 100 ng/ml of apicidin, and the inhibitory effect of apicidin was dose-dependent. The inhibition of BbHDAC3 by apicidin was confirmed by Western blot, IFAT, and reverse transcription-polymerase chain reaction (RT-PCR). Finally, apicidin potentially inhibited the in vitro growth of Babesia parasites. The lower IC(50) values of apicidin against apicomplexan parasites than those of mammalian cells point to HDAC as an excellent drug target. The findings of the present study indicate that BbHDAC3 is a potential target for apicidin and might be a promising target for the development of novel anti-babesial drugs.
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http://dx.doi.org/10.1016/j.vetpar.2012.06.026DOI Listing
December 2012

Apicoplast-targeting antibacterials inhibit the growth of Babesia parasites.

Antimicrob Agents Chemother 2012 Jun 5;56(6):3196-206. Epub 2012 Mar 5.

National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-Cho, Obihiro, Hokkaido, Japan.

The apicoplast housekeeping machinery, specifically apicoplast DNA replication, transcription, and translation, was targeted by ciprofloxacin, thiostrepton, and rifampin, respectively, in the in vitro cultures of four Babesia species. Furthermore, the in vivo effect of thiostrepton on the growth cycle of Babesia microti in BALB/c mice was evaluated. The drugs caused significant inhibition of growth from an initial parasitemia of 1% for Babesia bovis, with 50% inhibitory concentrations (IC(50)s) of 8.3, 11.5, 12, and 126.6 μM for ciprofloxacin, thiostrepton, rifampin, and clindamycin, respectively. The IC(50)s for the inhibition of Babesia bigemina growth were 15.8 μM for ciprofloxacin, 8.2 μM for thiostrepton, 8.3 μM for rifampin, and 206 μM for clindamycin. The IC(50)s for Babesia caballi were 2.7 μM for ciprofloxacin, 2.7 μM for thiostrepton, 4.7 μM for rifampin, and 4.7 μM for clindamycin. The IC(50)s for the inhibition of Babesia equi growth were 2.5 μM for ciprofloxacin, 6.4 μM for thiostrepton, 4.1 μM for rifampin, and 27.2 μM for clindamycin. Furthermore, an inhibitory effect was revealed for cultures with an initial parasitemia of either 10 or 7% for Babesia bovis or Babesia bigemina, respectively. The three inhibitors caused immediate death of Babesia bovis and Babesia equi. The inhibitory effects of ciprofloxacin, thiostrepton, and rifampin were confirmed by reverse transcription-PCR. Thiostrepton at a dose of 500 mg/kg of body weight resulted in 77.5% inhibition of Babesia microti growth in BALB/c mice. These results implicate the apicoplast as a potential chemotherapeutic target for babesiosis.
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http://dx.doi.org/10.1128/AAC.05488-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3370714PMC
June 2012

C-Terminal region of 48-kDa rhoptry protein for serological detection of Babesia caballi antibodies in horses.

Parasitol Int 2012 Sep 25;61(3):493-6. Epub 2012 Feb 25.

National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan.

A recombinant C-terminal antigen derived from Babesia caballi 48-kDa rhoptry protein (rBc48/CT) was made for the development of a serologically diagnostic test. Antiserum raised against the rBc48/CT reacted specifically with the corresponding native protein by Western blotting and the indirect fluorescent antibody test (IFAT). Next, an indirect enzyme-linked immunosorbent assay (Bc48/CT-ELISA) and an immunochromatographic test based on the Bc48/CT (Bc48/CT-ICT) were constructed and employed for the detection of an antibody to B. caballi in a variety of equine sera. The results of Bc48/CT-ELISA and Bc48/CT-ICT were highly concordant with those of IFAT and ELISA, with full-length protein of Bc48 used as the reference tests. Our results demonstrate the success of Bc48/CT as antigen for the serological diagnosis of B. caballi infection in horses.
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http://dx.doi.org/10.1016/j.parint.2012.02.006DOI Listing
September 2012

Phylogenetic relationships of Mongolian Babesia bovis isolates based on the merozoite surface antigen (MSA)-1, MSA-2b, and MSA-2c genes.

Vet Parasitol 2012 Mar 1;184(2-4):309-16. Epub 2011 Oct 1.

National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan.

We conducted a molecular epidemiological study on Babesia bovis in Mongolia. Three hundred blood samples collected from cattle grazed in seven different districts were initially screened using a previously established diagnostic polymerase chain reaction (PCR) assay for the detection of B. bovis-specific DNA. Positive samples were then used to amplify and sequence the hyper-variable regions of three B. bovis genes encoding the merozoite surface antigen (MSA)-1, MSA-2b, and MSA-2c. The diagnostic PCR assay detected B. bovis among cattle populations of all districts surveyed (4.4-26.0%). Sequences of each of the three genes were highly homologous among the Mongolian isolates, and found in a single phylogenetic cluster. In particular, a separate branch was formed only by the Mongolian isolates in the MSA-2b gene-based phylogenetic tree. Our findings indicate that effective preventative and control strategies are essential to control B. bovis infection in Mongolian cattle populations, and suggest that a careful approach must be adopted when using immunization techniques.
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http://dx.doi.org/10.1016/j.vetpar.2011.09.021DOI Listing
March 2012

Microbial communities associated with acetate-rich gas-petroleum reservoir surface facilities.

Biosci Biotechnol Biochem 2011 7;75(9):1835-7. Epub 2011 Sep 7.

Horonobe Research Institute for the Sub-Surface Environment, Northern Advancement Center for Science and Technology, Hokkaido.

We evaluated the microbial communities in acetate-rich production waters from separators of a high-temperature gas-petroleum reservoir in Higashi-Niigata, Japan. Bacterial and archaeal 16S rRNA gene libraries constructed from these waters were dominated by Acetobacterium-, Methanofollis-, and Methanosarcina-related sequences. The libraries constructed from enrichment cultures of the production waters were dominated by sequences related to the Acetobacterium- and Methanofollis-related sequences.
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http://dx.doi.org/10.1271/bbb.110243DOI Listing
January 2012

Molecular prevalence of different genotypes of Theileria orientalis detected from cattle and water buffaloes in Thailand.

J Parasitol 2011 Dec 14;97(6):1075-9. Epub 2011 Jun 14.

National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan.

Here we report on an epidemiological study regarding the molecular prevalence of different genotypes of Theileria orientalis present among domestic cattle and water buffalo populations bred in Thailand. A phylogenetic analysis based on the parasitic gene encoding a major piroplasm surface protein revealed the presence of 5 genotypes (Types 1, 3, 5, 7, and N-3) in cattle and 7 genotypes (Types 1, 3, 4, 5, 7, N-2, and N-3) in water buffaloes. Types 4, 7, and N-3 of T. orientalis were reported for the first time in water buffaloes. The previously reported C and Thai types from Thailand clustered as types 7 and 6, respectively, in the present analysis. Great similarities were observed among nucleotide sequences of isolates of the same genotype from cattle and water buffaloes, and, therefore, water buffaloes were considered to serve as a reservoir for these genotypes of T. orientalis in Thailand. In conclusion, T. orientalis parasites circulating in Thailand are more diverse in their genetic characters than previously anticipated.
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http://dx.doi.org/10.1645/GE-2846.1DOI Listing
December 2011

Molecular and serological prevalence of Babesia bovis and Babesia bigemina in water buffaloes in the northeast region of Thailand.

Vet Parasitol 2011 Jun 26;178(3-4):201-7. Epub 2011 Jan 26.

National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Nishi 2-13, Inada-cho, Obihiro, Hokkaido 080-8555, Japan.

Bovine babesiosis is a tick-transmitted hemoprotozoan disease that is mainly caused by Babesia bovis and Babesia bigemina and is characterized by significant morbidity and mortality worldwide. The disease is widespread in the northeastern region of Thailand, where an increasingly large part of the livestock is composed of water buffaloes. The present study was therefore conducted to investigate the epidemiological distribution of B. bovis and B. bigemina in water buffaloes in the northeastern region of Thailand. A total of 305 buffalo blood samples were randomly collected from five provinces and simultaneously analyzed by the nested PCR (nPCR) assay, ELISA, and IFAT techniques. The overall prevalence of B. bovis and B. bigemina was 11.2% and 3.6% by nPCR, 14.7% and 5.9% by ELISA, and 16.8% and 5.6% by IFAT, respectively. The high concordance between the molecular and the serological detection tests revealed the specificity and sensitivity of the diagnostic assays used for the detection of infection as well as the endemic stability status of the parasites in the surveyed areas. Statistically significant differences in the prevalence of the two infections were observed on the basis of age and location but not gender. Our data provide valuable information regarding the epidemiology of B. bovis and B. bigemina infection in water buffaloes in the northeastern region of Thailand which will likely be very beneficial for management and control programs of this disease.
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http://dx.doi.org/10.1016/j.vetpar.2011.01.041DOI Listing
June 2011

Toxoplasma gondii: a bradyzoite-specific DnaK-tetratricopeptide repeat (DnaK-TPR) protein interacts with p23 co-chaperone protein.

Exp Parasitol 2011 Apr 31;127(4):795-803. Epub 2011 Jan 31.

National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, 2-13 Inada-cho, Obihiro, Hokkaido 080-8555, Japan.

The DnaK-tetratricopeptide repeat (DnaK-TPR) gene (ToxoDB ID, TGME49_002020) is expressed predominantly at the bradyzoite stage. DnaK-TPR protein has a heat shock protein (DnaK) and tetratricopeptide repeat (TPR) domains with amino acid sequence similarity to the counterparts of other organisms (40.2-43.7% to DnaK domain and 41.1-66.0% to TPR domain). These findings allowed us to infer that DnaK-TPR protein is important in the tachyzoite-to-bradyzoite development or maintenance of cyst structure although the function of this gene is still unknown. An immunofluorescence assay (IFA) revealed that DnaK-TPR protein was expressed in Toxoplasma gondii-encysted and in vitro-induced bradyzoites and distributed in the whole part of parasite cells. We conducted yeast two-hybrid screening to identify proteins interacting with DnaK-TPR protein, and demonstrated that DnaK-TPR protein interacts with p23 co-chaperone protein (Tgp23). It was expected that DnaK-TPR protein would have a function as a molecular chaperon in bradyzoite cells associated with Tgp23. Possible mechanisms for this gene are discussed.
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http://dx.doi.org/10.1016/j.exppara.2011.01.015DOI Listing
April 2011

Molecular epidemiological survey of Theileria orientalis in Thua Thien Hue Province, Vietnam.

J Vet Med Sci 2011 May 24;73(5):701-5. Epub 2010 Dec 24.

National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, Japan.

Theileria orientalis is a benign bovine protozoan parasite that occasionally causes serious economic loss in the livestock industry. We report the findings of a molecular epidemiological survey of T. orientalis in 94 Vietnamese yellow cattle, 43 water buffaloes, 21 sheep, 21 goats and 85 blood-sucking ticks of cattle in the Thua Thien Hue province of Vietnam. The major piroplasm surface protein (MPSP) gene of T. orientalis was detected using polymerase chain reaction from 13 cattle (13.8%), 11 water buffaloes (25.6%), 1 sheep (4.8%) and 9 ticks (10.6%). Phylogenetic analysis using MPSP gene sequences showed the presence of seven genotypes, four previously categorized genotypes (Types 1, 3, 5 and 7) and three new genotypes (Types N-1, N-2 and N-3).
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http://dx.doi.org/10.1292/jvms.10-0472DOI Listing
May 2011

Spherical body protein 4 is a new serological antigen for global detection of Babesia bovis infection in cattle.

Clin Vaccine Immunol 2011 Feb 1;18(2):337-42. Epub 2010 Dec 1.

National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan.

Five Babesia bovis recombinant proteins, including merozoite surface antigen 2c (BbMSA-2c), C-terminal rhoptry-associated protein 1 (BbRAP-1/CT), truncated thrombospondin-related anonymous protein (BbTRAP-T), spherical body protein 1 (BbSBP-1), and spherical body protein 4 (BbSBP-4), were evaluated as diagnostic antigens to detect the infection in cattle. The recombinant proteins were highly antigenic when tested with experimentally B. bovis-infected bovine serum in Western blot analysis. Furthermore, five antisera that had been raised against each of the recombinant proteins reacted specifically with the corresponding authentic protein, as determined in Western blot analysis. Next, enzyme-linked immunosorbent assays (ELISAs) using these recombinant proteins were evaluated for diagnostic use, and the sensitivity and specificity of each protein were demonstrated with a series of serum samples from experimentally B. bovis-infected cattle. Furthermore, a total of 669 field serum samples collected from cattle in regions of B. bovis endemicity in seven countries were tested with the ELISAs, and the results were compared to those of an indirect fluorescent antibody test (IFAT), as a reference. Among five recombinant antigens, recombinant BbSBP-4 (rBbSBP-4) had the highest concordance rate (85.3%) and kappa value (0.705), indicating its reliability in the detection of specific antibodies to B. bovis in cattle, even in different geographical regions. Overall, we have successfully developed an ELISA based on rBbSBP-4 as a new serological antigen for a practical and sensitive test which will be applicable for epidemiologic survey and control programs in the future.
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http://dx.doi.org/10.1128/CVI.00388-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3067344PMC
February 2011

Genotypic diversity of Theileria orientalis detected from cattle grazing in Kumamoto and Okinawa prefectures of Japan.

J Vet Med Sci 2011 Mar 22;73(3):305-12. Epub 2010 Oct 22.

National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido.

Theileria orientalis is a benign protozoan species that is widely distributed in Japan, yet sometimes causes serious economic losses in the livestock industry. In this study, we conducted a molecular survey based on genes encoding the major piroplasm surface protein (MPSP) and p23 for T. orientalis detected in cattle grazing in southern areas of Japan, consisting of 2 farms in Kumamoto prefecture (Aso and Kuma districts) and 3 farms in Okinawa prefecture (Ishigaki, Iriomote, and Yonaguni Islands). High prevalence rates of T. orientalis infection were shown in all the cattle populations using the diagnostic MPSP- and p23-PCR assays. Phylogenetic analyses revealed 4 MPSP genotypes and 3 p23 genotypes. Furthermore, MPSP genotype-specific PCR methods were developed in this study and wide distributions of 5-district genotypes of T. orientalis were observed for the examined farms. Our results indicate that at least 5 types of T. orientalis exist in Kumamoto and Okinawa prefectures of Japan and that genotype-specific PCR assays are highly applicable for the quarantine of transported cattle and for epidemiological surveys of bovine theileriosis in Japan.
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http://dx.doi.org/10.1292/jvms.10-0263DOI Listing
March 2011

High-resolution characterization of Toxoplasma gondii transcriptome with a massive parallel sequencing method.

DNA Res 2010 Aug 3;17(4):233-43. Epub 2010 Jun 3.

1National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Japan.

For the last couple of years, a method that permits the collection of precise positional information of transcriptional start sites (TSSs) together with digital information of the gene-expression levels in a high-throughput manner was established. We applied this novel method, 'tss-seq', to elucidate the transcriptome of tachyzoites of the Toxoplasma gondii, which resulted in the identification of 124,000 TSSs, and they were clustered into 10,000 transcription regions (TRs) with a statistics-based analysis. The TRs and annotated ORFs were paired, resulting in the identification of 30% of the TRs and 40% of the ORFs without their counterparts, which predicted undiscovered genes and stage-specific transcriptions, respectively. The massive data for TSSs make it possible to execute the first systematic analysis of the T. gondii core promoter structure, and the information showed that T. gondii utilized an initiator-like motif for their transcription in the major and novel motif, the downstream thymidine cluster, which was similar to the Y patch observed in plants. This encyclopaedic analysis also suggested that the TATA box, and the other well-known core promoter elements were hardly utilized.
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http://dx.doi.org/10.1093/dnares/dsq013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2920756PMC
August 2010

Toxoplasma gondii deoxyribose phosphate aldolase-like protein (TgDPA) interacts with actin depolymerizing factor (TgADF) to enhance the actin filament dynamics in the bradyzoite stage.

Mol Biochem Parasitol 2010 Sep 28;173(1):39-42. Epub 2010 Apr 28.

National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, Japan.

The Toxoplasma gondii deoxyribose phosphate aldolase-like (TgDPA) gene is expressed predominantly in bradyzoites. This finding allowed us to infer that TgDPA is important in the tachyzoite-to-bradyzoite development or maintenance of cyst structure although the function of this gene is still unknown. We conducted yeast two-hybrid screening to identify proteins interacting with TgDPA, and the actin depolymerizing factor (TgADF) gene was obtained. Co-immunoprecipitation and a GST pull-down assay demonstrated that TgDPA interacts with TgADF. To reveal the significance of the protein-protein interaction between TgDPA and TgADF, actin polymerization and disassembly kinetics were examined. Addition of GST-TgDPA to TgADF lowered the extent of actin polymerization and enhanced the filamentous actin disassembly. These results demonstrated that this is the novel protein-protein interaction in T. gondii, and that TgDPA can enhance the activity of TgADF. This phenomenon might play an important role in T. gondii bradyzoites by affecting the actin turnover.
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http://dx.doi.org/10.1016/j.molbiopara.2010.04.007DOI Listing
September 2010

pfaB products determine the molecular species produced in bacterial polyunsaturated fatty acid biosynthesis.

FEMS Microbiol Lett 2009 Jun 21;295(2):170-6. Epub 2009 Apr 21.

Laboratory of Environmental Molecular Biology, Faculty of Environmental Earth Science, Hokkaido University, Sapporo, Japan.

When pDHA4, a vector carrying all five pfaA-pfaE genes responsible for docosahexaenoic acid (DHA; 22:6) biosynthesis in Moritella marina MP-1, was coexpressed in Escherichia coli with the individual pfaA-pfaD genes for eicosapentaenoic acid (EPA; 20:5) biosynthesis from Shewanella pneumatophori SCRC-2738, both polyunsaturated fatty acids were synthesized only in the recombinant carrying pfaB for EPA synthesis. Escherichia coli coexpressing a deleted construct comprising pfaA, pfaC, pfaD and pfaE for EPA and pfaB for DHA produced EPA and DHA. Both EPA and DHA were detected in bacteria that inherently contained pfa genes for DHA. These results suggest that PfaB is the key enzyme determining the final product in EPA or DHA biosynthesis.
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http://dx.doi.org/10.1111/j.1574-6968.2009.01582.xDOI Listing
June 2009

High seroprevalence of Encephalitozoon cuniculi in pet rabbits in Japan.

J Vet Med Sci 2008 Dec;70(12):1301-4

National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Hokkaido, Japan.

Infection with Encephalitozoon cuniculi in rabbits frequently exists as a chronic, latent infection, and only a percentage of infected animals develop clinical disease. This study presents a seroepidemiological study of E. cunicucli infection in 337 pet rabbits collected from 20 prefectures in Japan in 2006 and 2007, using enzyme-linked immunosorbent assay (ELISA) capable of measuring IgG and IgM antibodies. These rabbits were divided into the following four groups: healthy and isolated rabbits (n=74, group I), healthy and companioned rabbits (n=121, group II), neurologically diseased rabbits (n=105, group III), and other diseased rabbits (n=37, group IV). Using ELISA for IgG antibodies, the highest detection rate, 81%, was seen in group III, the second highest, 75.2%, in group II, and the lowest, 29.7%, in group I, which was significantly different to the other groups except for group IV (43.2%). On the other hand, when ELISA was used for IgM antibody detection, 14-40% of rabbits in the four groups were also observed to have anti-E. cuniculi IgM. This study demonstrated high seroprevalence of E. cuniculi in not only neurologically diseased rabbits but also healthy and other diseased rabbits.
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http://dx.doi.org/10.1292/jvms.70.1301DOI Listing
December 2008

Toxoplasma gondii: Identification and characterization of bradyzoite-specific deoxyribose phosphate aldolase-like gene (TgDPA).

Exp Parasitol 2009 Jan 10;121(1):55-63. Epub 2008 Oct 10.

National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, 2-13 Inada-cho, Obihiro, Hokkaido 080-8555, Japan.

Toxoplasma gondii undergoes stage conversion from tachyzoites to bradyzoites in intermediate hosts. There have been many reports on bradyzoite-specific genes which are thought to be involved in stage conversion. Here, we described a novel T. gondii deoxyribose phosphate aldolase-like gene (TgDPA) expressing predominantly in bradyzoites. The TgDPA gene encodes 286 amino acids having a predicted molecular weight of 31kDa. Sequence analysis revealed that TgDPA had a deoxyribose phosphate aldolase (DeoC) domain with about 30% homology with its Escherichia coli counterpart. RT- and quantitative PCR analyses showed that the TgDPA gene was more expressed in bradyzoites and that its expression gradually increased during in vitro tachyzoite-to-bradyzoite stage conversion. A polyclonal antibody against recombinant TgDPA protein was raised in rabbits, and immunofluorescent analysis demonstrated that TgDPA was expressed in bradyzoites in vivo and in vitro. These findings indicate that the TgDPA gene is a new bradyzoite-specific marker and might play a role in bradyzoites.
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http://dx.doi.org/10.1016/j.exppara.2008.09.018DOI Listing
January 2009

Molecular and biochemical characterization of Toxoplasma gondii beta-hydroxyacyl-acyl carrier protein dehydratase (FABZ).

Parasitol Res 2008 May 15;102(6):1301-9. Epub 2008 Feb 15.

National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, 2-13 Inada-cho, Obihiro, Hokkaido 080-8555, Japan.

Toxoplasma gondii, unlike its mammalian host, utilizes a type II fatty acid biosynthesis pathway in which the steps of fatty acid biosynthesis are catalyzed by independent enzymes. Due to this difference, the enzymes of this pathway are good targets for the development of new therapeutic drugs directed against toxoplasmosis. In this report, we show by using reverse transcription-polymerase chain reaction analysis that beta-Hydroxyacyl-acyl carrier protein dehydratase (TgFABZ) is expressed both in tachyzoites and bradyzoites. Indirect immunofluorescence antibody test further shows the localization of TgFABZ protein in the apicoplast of both tachyzoites and bradyzoites. Enzyme dynamic analysis shows that the purified recombinant TgFABZ protein is soluble and active. The Km value of the enzyme for its substrate analog crotonoyl-CoA was estimated to be 82.57 +/- 10 microM.
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http://dx.doi.org/10.1007/s00436-008-0909-4DOI Listing
May 2008