PubMed Health⌕ Search

Biomedical subjects

Seiki Kobayashi

Publications and source records attributed to Seiki Kobayashi.

At least 19 recordsLinked to original sources
PubMed2006

Genetic diversity of glucose phosphate isomerase from Entamoeba histolytica.

To investigate the molecular basis of zymodeme analysis in the enteric protozoan parasite Entamoeba histolytica, genes encoding glucose phosphate isomerase (GPI) were isolated from four representative E. histolytica strains belonging to zymodeme II, IIalpha-, XIV, or XIX. Two alleles were obtained from each strain; six alleles with eight polymorphic nucleotide positions were identified among the four strains. Two of these eight polymorphic nucleotides resulted in non-conserved amino acid substitutions. Three GPI isoenzymes with distinct predicted isoelectric points were identified, which agrees well with the observed electrophoretic patterns of GPI from these strains. Amino acid comparisons of GPI from E. histolytica and other organisms revealed that all amino acid residues implicated for substrate binding and catalysis were conserved. Biochemical characterization of recombinant E. histolytica GPI confirmed that it possessed kinetic parameters similar to GPI from other organisms. The electrophoretic mobility of three GPI isoenzymes was examined by starch gel electrophoresis. Thus, we have established the molecular basis of the classical isoenzymes patterns that have been used for grouping E. histolytica isolates and for differentiation of E. histolytica from non-pathogenic Entamoeba dispar.

Amino Acid Sequence↗
PubMed2006

Entamoeba histolytica: differences in phagosome acidification and degradation between attenuated and virulent strains.

Phagocytosis is the important virulent determinant of the enteric protozoan parasite Entamoeba histolytica. We compared the kinetics of phagosome maturation of attenuated and highly-virulent strains of E. histolytica using video microscopy. Phagosomes of attenuated strains were acidified rapidly within 2 min after phagosome formation (at the rate of 0.96 pH/min), persisted at pH 4.46+/-0.13, and degraded ingested GFP-Leishmania very efficiently (90-94% GFP fluorescence was lost in 30 min), while phagosomes of highly-virulent strains were acidified slowly (0.69 pH/min), persisted at 5.11+/-0.23, and degraded GFP less efficiently (60-71% decrease). These results suggest that efficiency of phagosome maturation is most probably inversely correlated with apparent virulence.

Animals↗
PubMed2006

Effect of artificial gastrointestinal fluids on the excystation and metacystic development of Entamoeba invadens.

The effect of artificial gastric fluid (AGF), containing 0.5% pepsin and 0.6% hydrochloric acid, pH 1.8, in distilled water, on the excystation and metacystic development of Entamoeba invadens was examined. Excystation, which was assessed by counting the number of metacystic amoebae after inducing excystation, was enhanced by pretreatment of cysts with AGF for 30 to 60 min at 37 degrees C but not 26 degrees C. Longer exposure of cysts to AGF significantly reduced their viability. Significant enhancement of excystation was observed by pretreatment of cysts with distilled water only at 37 degrees C. In addition, 0.6% hydrochloric acid had a comparable enhancing effect on excystation to AGF. Metacystic development, when determined by the number of nuclei in amoeba, was slightly enhanced by pretreatment with AGF. An artificial intestinal fluid (AIF), containing 1% pancreatin, 1% sodium bicarbonate, and 5% ox bile, pH 8.0, in distilled water, had a significant toxic effect on cysts, where 1% pancreatin had neither an enhancing effect on excystation nor a toxic effect on cysts, whereas 5% ox bile had a toxic effect on cysts. Pretreatment of cysts with AGF followed by AIF had a similar toxic effect on cysts to that by AIF only. These results suggest that gastric fluid but not intestinal fluid at 37 degrees C contributes to enhancing excystation for Entamoeba infection.

Animals↗
PubMed2006

Diversity of clinical isolates of Entamoeba histolytica in Japan.

In Japan, amebiasis is domestically transmitted by two major populations: male homosexuals and mentally handicapped persons, which is remarkably different from most other developed countries where Entamoeba dispar infection is predominantly observed. Here we briefly summarize epidemiology of amebiasis in Japan. We also review our current understanding of the diversity of Entamoeba histolytica clinical isolates in Japan, based on polymorphic genetic markers, clinical representations, and in vivo virulence, using an animal model.

Animals↗
PubMed2005

Differences in morphology of phagosomes and kinetics of acidification and degradation in phagosomes between the pathogenic Entamoeba histolytica and the non-pathogenic Entamoeba dispar.

Phagocytosis plays an important role in the pathogenicity of the intestinal protozoan parasite Entamoeba histolytica. We compared the morphology of phagosomes and the kinetics of phagosome maturation using conventional light and electron microscopy and live imaging with video microscopy between the virulent E. histolytica and the closely-related, but non-virulent E. dispar species. Electron micrographs showed that axenically cultivated trophozoites of the two Entamoeba species revealed morphological differences in the number of bacteria contained in a single phagosome and the size of phagosomes. Video microscopy using pH-sensitive fluorescein isothiocynate-conjugated yeasts showed that phagosome acidification occurs within 2 min and persists for >12 h in both species. The acidity of phagosomes significantly differed between two species (4.58 +/- 0.36 or 5.83 +/- 0.38 in E. histolytica or E. dispar, respectively), which correlated well with the differences in the kinetics of degradation of promastigotes of GFP-expressing Leishmania amazonensis. The acidification of phagosomes was significantly inhibited by a myosin inhibitor, whereas it was only marginally inhibited by microtubules or actin inhibitors. A specific inhibitor of vacuolar ATPase, concanamycin A, interrupted both the acidification and degradation in phagosomes in both species, suggesting the ubiquitous role of vacuolar ATPase in the acidification and degradation in Entamoeba. In contrast, inhibitors against microtubules or cysteine proteases (CP) showed distinct effects on degradation in phagosomes between these two species. Although depolymerization of microtubules severely inhibited degradation in phagosomes of E. histolytica, it did not affect degradation in E. dispar. Similarly, the inhibition of CP significantly reduced degradation in phagosomes of E. histolytica, but not in E. dispar. These data suggest the presence of biochemical or functional differences in the involvement of microtubules and proteases in phagosome maturation and degradation between the two species.

Actins↗
PubMed2005

Isolation and functional analysis of a chk2 homologue from Entamoeba histolytica.

Mammalian Chk2 is a Ser/Thr kinase required for cell-division arrest induced by DNA damage. We found six new kinase genes of Entamoeba histolytica by analysis in silico. One of the kinase genes was a homologue of human chk2 gene. The chk2 homologue gene (Eh chk2) was expected to encode 398 amino acids and showed nearly 50% homology to human Chk2 in amino acid sequence. Eh chk2 had a catalytic domain of Ser/Thr kinase and a fork head-associated (FHA) domain that is highly conserved among Chk2 homologues in vertebrates. To examine the biological functions of Eh chk2, we synthesized Eh chk2 mRNA in vitro and injected it into immature frog eggs (Xenopus laevis oocytes) as a model system of cell division. Eh chk2 markedly delayed the cell division of frog eggs by disrupting transition of G2 phase to M phase. Eh chk2 also inhibited the activation of p42 MAPK and Cdc2 kinase which are representative events induced by cell division. These results suggest that Eh chk2 gene should be a cell-division regulator in E. histolytica.

Amino Acid Sequence↗
PubMed2005

Immune responses against a single CD8+-T-cell epitope induced by virus vector vaccination can successfully control Trypanosoma cruzi infection.

In order to develop CD8+-T-cell-mediated immunotherapy against intracellular infectious agents, vaccination using recombinant virus vectors has become a promising strategy. In this study, we generated recombinant adenoviral and vaccinia virus vectors expressing a single CD8+-T-cell epitope, ANYNFTLV, which is derived from a Trypanosoma cruzi antigen. Immunogenicity of these two recombinant virus vectors was confirmed by the detection of ANYNFTLV-specific CD8+ T cells in the spleens of immunized mice. Priming/boosting immunization using combinations of these two recombinant virus vectors revealed that the adenovirus vector was efficient for priming and the vaccinia virus vector was effective for boosting the CD8+-T-cell responses. Moreover, we also demonstrated that the ANYNFTLV-specific CD8+-T-cell responses were further augmented by coadministration of recombinant vaccinia virus vector expressing the receptor activator of NFkappaB (RANK) ligand as an adjuvant. By priming with the adenovirus vector expressing ANYNFTLV and boosting with the vaccinia virus vectors expressing ANYNFTLV and RANK ligand, the immunized mice were efficiently protected from subsequent challenge with lethal doses of T. cruzi. These results indicated, for the first time, that the induction of immune responses against a single CD8+-T-cell epitope derived from an intrinsic T. cruzi antigen was sufficient to control lethal T. cruzi infection.

Adenoviridae↗
PubMed2005

Axenic cultivation of Entamoeba dispar in newly designed yeast extract-iron-gluconic acid-dihydroxyacetone-serum medium.

Yeast extract-iron-gluconic acid-dihydroxyacetone-serum medium that allows axenic cultivation of Entamoeba dispar was designed based on casein-free yeast extract-iron-serum (YI-S) medium, and the usefulness of the medium was assessed. The main differences from YI-S medium are replacement of glucose by gluconic acid, addition of dihydroxyacetone and D-galacturonic acid monohydrate, and sterilization by filtration. This medium promoted the axenic growth of 5 strains of E. dispar (2 strains of nonhuman primate isolates and 3 strains of human isolates). In addition, to clarify the biological basis for the growth of E. dispar in this medium, analyses of relevant enzymes on the glycolytic pathway of the amoebae as well as of the protozoans that are the best culture supplement for amoebae are being performed.

Animals↗
PubMed2004

Entamoeba invadens: cysteine protease inhibitors block excystation and metacystic development.

We examined the effects of six cysteine protease inhibitors on the excystation and metacystic development of Entamoeba invadens. Excystation, which was assessed by counting the number of metacystic amoebae after the induction of excystation, was inhibited by the cysteine protease inhibitors Z-Phe-Ala-DMK and E-64d in a concentration-dependent manner during incubation compared to the controls. Neither inhibitor had a significant effect on cyst viability; thus, their inhibitory effects were not due to the toxic effect on cysts. Metacystic development, when determined by the number of nuclei in amoeba, was also inhibited by these protease inhibitors, because the percentage of 4-nucleate amoebae was higher than in the controls on Day 3 of incubation. Although other cysteine protease inhibitors, Z-Phe-Phe-DMK, E-64, ALLM, and cathepsin inhibitor III, had a weak or little effect on the excystation, they inhibited cysteine protease activity in the lysates of E. invadens cysts. Broad bands with gelatinase activity of metacystic amoebae, as well as cysts and trophozoites, were detected in the gelatin substrate gel electrophores and were inhibited by Z-Phe-Ala-DMK. There was a difference in the protease composition between cysts and trophozoites, and the protease composition of metacystic amoebae changed from cyst-type to trophozoite-type during development. These results strongly suggest that cysteine proteases contribute to the excystation and metacystic development of E. invadens, which leads to successful infection.

Animals↗
PubMed2004

Different effects of cytochalasins on the growth and differentiation of Entamoeba invadens.

The effect of five different cytochalasins on the growth, encystation and excystation of Entamoeba invadens was examined. At 10 microM, cytochalasins B, D, E and dihydrocytochalasin B markedly inhibited growth. Encystation was inhibited by cytochalasin D at 1 microM but not by other cytochalasins at the same concentration, whereas it was inhibited by 10 microM of cytochalasins B, E and dihydrocytochalasin B as well as cytochalasin D. Excystation, which was assessed by counting the number of metacystic amoebae after inducing excystation, was markedly enhanced by cytochalasin D as previously demonstrated, whereas the enhancing effect of cytochalasins A, B and dihydrocytochalasin B was weak. In contrast, cytochalasin E at 10 microM inhibited excystation and metacystic development. These results indicate that there is a difference in the effect of different cytochalasins on the growth and differentiation of E. invadens, depending on differences in their chemical structure.

Animals↗
PubMed2004

Effects of bisphosphonates on the growth of Entamoeba histolytica and Plasmodium species in vitro and in vivo.

The effects of a series of 102 bisphosphonates on the inhibition of growth of Entamoeba histolytica and Plasmodium falciparum in vitro have been determined, and selected compounds were further investigated for their in vivo activity. Forty-seven compounds tested were active (IC(50) < 200 microM) versus E. histolytica growth in vitro. The most active compounds (IC(50) approximately 4-9 microM) were nitrogen-containing bisphosphonates with relatively large aromatic side chains. Simple n-alkyl-1-hydroxy-1,1-bisphosphonates, known inhibitors of the enzyme farnesylpyrophosphate (FPP) synthase, were also active, with optimal activity being found with C9-C10 side chains. However, numerous other nitrogen-containing bisphosphonates known to be potent FPP synthase inhibitors, such as risedronate or pamidronate, had little or no activity. Several pyridine-derived bisphosphonates were quite active (IC(50) approximately 10-20 microM), and this activity was shown to correlate with the basicity of the aromatic group, with activity decreasing with increasing pK(a) values. The activities of all compounds were tested versus a human nasopharyngeal carcinoma (KB) cell line to enable an estimate of the therapeutic index (TI). Five bisphosphonates were selected and then screened for their ability to delay the development of amebic liver abscess formation in an E. histolytica infected hamster model. Two compounds were found to decrease liver abscess formation at 10 mg/kg ip with little or no effect on normal liver mass. With P. falciparum, 35 compounds had IC(50) values <200 microM in an in vitro assay. The most active compounds were also simple n-alkyl-1-hydroxy-1,1-bisphosphonates, having IC(50) values around 1 microM. Five compounds were again selected for in vivo investigation in a Plasmodium berghei ANKA BALB/c mouse suppressive test. The most active compound, a C9 n-alkyl side chain containing bisphosphonate, caused an 80% reduction in parasitemia with no overt toxicity. Taken together, these results show that bisphosphonates appear to be useful lead compounds for the development of novel antiamebic and antimalarial drugs.

Animals↗
PubMed2003

Cutting edge: a potent adjuvant effect of ligand to receptor activator of NF-kappa B gene for inducing antigen-specific CD8+ T cell response by DNA and viral vector vaccination.

The ligand to receptor activator of NF-kappaB (RANK-L)/RANK interaction has been implicated in CD40 ligand/CD40-independent T cell priming by dendritic cells. In this report, we show that the coadministration of the RANK-L gene with a Trypanosoma cruzi gene markedly enhances the induction of Trypanosoma Ag-specific CD8(+) T cells and improves the DNA vaccine efficacy. A similarly potent adjuvant effect of the RANK-L gene on the induction of Ag-specific CD8(+) T cells was also observed when recombinant influenza virus expressing murine malaria Ag was used as an immunogen. In contrast, the coadministration of the CD40L gene was not effective in these systems. Our results demonstrated, for the first time, the potent immunostimulatory effect of the RANK-L gene to improve the CD8(+) T cell-mediated immunity against infectious agents.

Adjuvants, Immunologic↗
PubMed2003

Involvement of ICOS-B7RP-1 costimulatory pathway in the regulation of immune responses to Leishmania major and Nippostrongylus brasiliensis infections.

The ICOS-B7RP-1-mediated T cell costimulatory pathway has been implicated crucial for T cell activation and differentiation. In this study, we investigated the role of this costimulation in the regulation of immune responses to parasitic infections by using blocking antibody against B7RP-1 as well as ICOS-deficient mice. The administration of anti-B7RP-1 monoclonal antibody (mAb) significantly suppressed the footpad swelling in susceptible BALB/c mice upon Leishmania major infection. The observation was consistent not only with the significant suppression of IL-4, IL-5 and IL-10 secretion from lymph node cells, which were derived from L. major-infected mice, but also with the significant reduction of total serum IgE and IgG(1) in anti-B7RP-1 mAb-treated BALB/c mice. Infection of ICOS-deficient mice with L. major also suggested the impaired Th2 immune responses in the absence of this costimulation. The immunological function of ICOS-B7RP-1 costimulatory pathway in infection was further confirmed by infecting anti-B7RP-1 mAb-treated wild type or ICOS-deficient mice with Nippostrongylus brasiliensis. The characteristic elevation of total serum IgE and eosinophilia upon N. brasiliensis infection was suppressed by blocking this costimulation. Moreover, the protection to N. brasiliensis adult worms was suppressed in anti-B7RP-1 mAb-treated wild type or ICOS-deficient mice. These results suggest the crucial role of this costimulatory pathway in the regulation of Th2-biased T cell differentiation and in host immune responses against L. major and N. brasiliensis infections.

Animals↗
PubMed2003

Involvement of signaling through protein kinase C and phosphatidylinositol 3-kinase in the excystation and metacystic development of Entamoeba invadens.

Using an axenic excystation system in vitro, we examined the effect of protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K), which are signaling molecules responsible for numerous cellular responses, on the excystation and metacystic development of Entamoeba invadens. Excystation, which was assessed by counting the number of metacystic amoebae after the induction of excystation, was inhibited by the PKC inhibitors staurosporine, chelerythrine chloride and calphostin C in a concentration-dependent manner during incubation, compared with the controls. As cyst viability was not affected by these inhibitors, reduced excystation was not due to their direct toxic effects on cysts. Metacystic development, when determined by the number of nuclei in the amoebae, was delayed by these PKC inhibitors, because the percentage of 1-nucleate amoebae was lower than in controls at day 3 of incubation. Wortmannin, a potent inhibitor of PI3K, also inhibited excystation and metacystic development of E. invadens in a concentration-dependent manner, compared with the controls. These results indicate that signaling through PKC and PI3K contributes to the excystation and metacystic development of E. invadens.

Androstadienes↗
PubMed2003

Identification and characterization of two isoenzymes of methionine gamma-lyase from Entamoeba histolytica: a key enzyme of sulfur-amino acid degradation in an anaerobic parasitic protist that lacks forward and reverse trans-sulfuration pathways.

To better understand the metabolism of sulfur-containing amino acids, which likely plays a key role in a variety of cell functions, in Entamoeba histolytica, we searched the genome data base for genes encoding putative orthologs of enzymes known to be involved in the metabolism. The search revealed that E. histolytica possesses only incomplete cysteine-methionine conversion pathways in both directions. Instead, this parasite possesses genes encoding two isoenzymes of methionine gamma-lyase (EC 4.4.1.11, EhMGL1/2), which has been implicated in the degradation of sulfur-containing amino acids. The two amebic MGL isoenzymes, showing 69% identity to each other, encode 389- and 392-amino acid polypeptides with predicted molecular masses of 42.3 and 42.7 kDa and pIs of 6.01 and 6.63, respectively. Amino acid comparison and phylogenetic analysis suggested that these amebic MGLs are likely to have been horizontally transferred from the Archaea, whereas an MGL from another anaerobic protist Trichomonas vaginalis has MGL isotypes that share a common ancestor with bacteria. Enzymological and immunoblot analyses of the partially purified native amebic MGL confirmed that both of the MGL isotypes are expressed in a comparable amount predominantly in the cytosol and form a homotetramer. Recombinant EhMGL1 and 2 proteins catalyzed degradation of L-methionine, DL-homocysteine, L-cysteine, and O-acetyl-L-serine to form alpha-keto acid, ammonia, and hydrogen sulfide or methanethiol, whereas activity toward cystathionine was negligible. These two isoenzymes showed notable differences in substrate specificity and pH optimum. In addition, we showed that EhMGL is an ideal target for the development of new chemotherapeutic agents against amebiasis by demonstrating an amebicidal effect of the methionine analog trifluoromethionine on trophozoites in culture (IC50 18 mum) and that this effect of trifluoromethionine was completely abolished by the addition of the MGL-specific inhibitor DL-propargylglycine.

Amino Acids, Sulfur↗
PubMed2003

Structural properties of dibenzosuberanylpiperazine derivatives for efficient reversal of chloroquine resistance in Plasmodium chabaudi.

For the purpose of developing chemosensitizers to reverse chloroquine (CQ) resistance in Plasmodium chabaudi in vivo, dibenzosuberanylpiperazine (1-(10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5-yl)piperazine) (DSP) and its piperazin-1-yl derivatives were synthesized systematically. DSP hydrochloride (3) was obtained from the reaction of dibenzosuberanyl chloride with piperazine in the presence of 1,8-diazabicyclo[5,4,0]-7-undecene (DBU). To understand the relationship between the substituent patterns of DSP derivatives and their biological activities, 13 hydroxyalkyl or hydroxyalkenyl derivatives were synthesized by an attack of the piperazine secondary amine of 3 on commercially available epoxides in the presence of triethylamine or DBU, and three alkyl or alkynyl derivatives were synthesized by the reactions of 3 with the corresponding organic chlorides in the presence of DBU. In both reactions, the yield was a maximum of 90%. The biological activities of the synthesized compounds were evaluated on the basis of two values: antimalarial activity and reversal activity. The values of antimalarial activities by single administration of 17 test compounds were not effective, being in the range 67-152% on day 4 after infection of Plasmodium chabaudi to mice except for the administration of 3-(dibenzosuberanylpiperazin-1-yl)-1-butene (29, 22%). On the other hand, administration of the seven test compounds (50 mg/kg dose) combined with CQ (3-4 mg/kg) gave high reversal activities, namely, low values (0% on day 4). The effective test compounds were those obtained by introducing the following substituents: 2-hydroxybutyl (24), 2-hydroxyhexen-5-yl (27), 2-hydroxybuten-3-yl (28a), 2-substituted 1-hydroxybuten-3-yl (28b), 4-acetoxybutyn-2-yl (30), 4-hydroxybutyn-2-yl (31), and 3-substituted buten-1-yl (29), which correspond to the nonbulky groups of hydroxyalkyl (C4), hydroxyalkenyl (C4-C6), hydroxyalkynyl (C4), or alkenyl (C4). These results may lead to the development of an approach to developing clinically applicable chemosensitizers for drug-resistant malaria.

Animals↗
PubMed2003

Induction of permeability changes and death of vertebrate cells is modulated by the virulence of Entamoeba spp. isolates.

Although Entamoeba histolytica is capable of inducing an apoptotic response in vertebrate cells in vitro (Cell. Microbiol. 2 (2000) 617), it is not known whether vertebrate cell death requires direct amoeba-vertebrate cell contact or simply the presence of amoebae in the area of the vertebrate cells. In addition, Entamoeba spp. vary in their virulence and pathogenicity. The potential effects of these critical parameters also have not been elucidated. We tested the virulent HM-1:IMSS isolate and the non-virulent Rahman isolate of E. histolytica, and the non-virulent E. dispar CYNO16:TPC isolate against two vertebrate cell lines, HeLa and Chinese hamster ovary cells in vitro using ethidium homodimer as a fluorescent indicator of changes in vertebrate cell permeability. Fluorescence appeared in vertebrate cell nuclei within approximately 2-3 min of contact between HM-1 amoebae and vertebrate cells independent of vertebrate cell type. However, vertebrate cells in the immediate vicinity of but not contacted by HM-1 amoebae were not affected. In contrast, although both E. histolytica Rahman and E. dispar CYNO16 amoebae moved freely among and contacted vertebrate cells, the nuclei of the vertebrate cells never fluoresced implying that the cells remained alive and impermeant to the ethidium homodimer. This is the first demonstration that direct contact between virulent amoebae and vertebrate cells is required to kill vertebrate cells and that the process is restricted to virulent Entamoeba isolates. An understanding at the molecular level of the processes involved could help to reduce the pathology associated with this parasite.

Animals↗