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Asao Makioka

Publications and source records attributed to Asao Makioka.

12 recordsLinked to original sources
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↗
PubMed2005

Characterization of protein geranylgeranyltransferase I from the enteric protist Entamoeba histolytica.

Entamoeba histolytica is a unique protozoan parasite possessing both protein farnesyltransferase and geranylgeranyltrasferase I (GGT-I) for isoprenylation of small GTPases. In this study, we demonstrated unique enzymological properties of the amebic GGT-I (EhGGT-I), including substrate specificity and insensitivity to known mammalian inhibitors. Some of important residues of the catalytic beta subunit implicated in the specificity for GTPase acceptors and prenyl donors are substituted in EhGGT-I. Recombinant alpha and beta subunits of EhGGT-I, co-expressed in Escherichia coli, showed activity to transfer geranylgeranyl to both human wild-type (CVLS) and mutant (CVLL) H-Ras, while the mammalian GGT-I geranylgeranylated, but not farnesylated, only mutant H-Ras. All the representative amebic Ras and Rho/Rac small GTPases with phenylalanine, leucine, methionine, or alanine terminus were preferentially geranylgeranylated by EhGGT-I. This indicates that the acceptor specificity of the amebic GGT-I is remarkably broader than that of its mammalian counterpart. In contrast to EhFT, which farnesylates but not geranylgeranlylates solely EhRas4-CVVA, EhGGT-I also showed significant farnesyltransferase activity against Ras GTPase acceptors. EhGGT-I showed remarkable resistance to peptidomimetics known to inhibit mammalian GGT-I. Together with our previous observation that this parasite does not appear to depend on farnesylation for a majority of Ras and Rho/Rac, these data indicate that biological and biochemical advantages leading to the evolutional selection of this isoprenyl modification must exist uniquely in this parasitic protist. Finally, remarkable biochemical differences in binding to substrates and inhibitors between amebic and mammalian GGT-I highlight this enzyme as an attractive target for the development of new chemotherapeutics against amebiasis.

Alkyl and Aryl Transferases↗
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↗
PubMed2003

Molecular cloning and characterization of a protein farnesyltransferase from the enteric protozoan parasite Entamoeba histolytica.

Genes encoding alpha- and beta-subunits of a putative protein farnesyltransferase (FT) from the enteric protozoan parasite Entamoeba histolytica were obtained and their biochemical properties were characterized. Deduced amino acid sequences of the alpha- and beta-subunit of E. histolytica FT (EhFT) were 298- and 375-residues long with a molecular mass of 35.6 and 42.6 kDa, and a pI of 5.43 and 5.65, respectively. They showed 24% to 36% identity to and shared common signature domains and repeats with those from other organisms. Recombinant alpha- and beta-subunits, co-expressed in Escherichia coli, formed a heterodimer and showed activity to transfer farnesyl using farnesylpyrophosphate as a donor to human H-Ras possessing a C-terminal CVLS, but not a mutant H-Ras possessing CVLL. Among a number of small GTPases that belong to the Ras superfamily from this parasite, we identified EhRas4, which possesses CVVA at the C terminus, as a sole farnesyl acceptor for EhFT. This is in contrast to mammalian FT, which utilizes a variety of small GTPases that possess a C-terminal CaaX motif, where X is serine, methionine, glutamine, cysteine, or alanine. EhFT also showed remarkable resistance against a variety of known inhibitors of mammalian FT. These results suggest that remarkable biochemical differences in binding to substrates and inhibitors exist between amebic and mammalian FTs, which highlights this enzyme as a novel target for the development of new chemotherapeutics against amebiasis.

Alkyl and Aryl Transferases↗
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↗
PubMed2002

Effect of jasplakinolide on the growth, invasion, and actin cytoskeleton of Plasmodium falciparum.

The effect of jasplakinolide (JAS), an actin-polymerizing and filament-stabilizing drug, on the growth, invasion, and actin cytoskeleton of Plasmodium falciparum was examined. Jasplakinolide markedly decreased the parasitemia in a synchronized culture of P. falciparum strain FCR-3 in a time- and concentration-dependent manner. The decrease became evident at day 2 at concentrations of 0.3 micro M and above, and parasites finally disappeared at day 4. Giemsa-stained smears of P. falciparum-infected erythrocytes demonstrated that there was no effect on the development of schizonts from ring forms. Merozoites were released from the infected erythrocytes in a normal manner with and without JAS. However, there were no ring form-infected erythrocytes when JAS was administered, even after the release of merozoites. This indicates that the merozoites exposed to JAS failed to invade erythrocytes. The inhibitory effect of JAS on the parasitemia was reversed by the removal of the drug after exposure to 1 micro M of JAS for 1 day. Electron microscopy revealed that the merozoites treated with JAS showed a protrusion of the apical end which contained the microfilament structure. Immunoblot analysis indicated that the JAS treatment increased F-actin filaments of merozoites but had no effect on those of the trophozoites and schizonts. Therefore, this study demonstrated that JAS has an antimalarial activity.

Actin Cytoskeleton↗
PubMed2002

Possible role of calcium ions, calcium channels and calmodulin in excystation and metacystic development of Entamoeba invadens.

The effect of calcium ions (Ca(2+)) and calmodulin (CaM) on the excystation and metacystic development of Entamoeba invadens was examined by transfer of cysts to a growth medium containing calcium antagonists and CaM inhibitors. Excystation, which was assessed by counting the number of metacystic amoebae after induction of excystation, was inhibited by the calcium chelators ethyleneglycol bis (beta-aminoethyl ether)- N,N'-tetraacetate (EGTA) and ethylene-diaminetetraacetate (EDTA), with EDTA being more potent than EGTA. The inhibitory effect of higher concentrations of these chelators on excystation was associated with reduced viability of cysts. Metacystic development, when determined by the number of nuclei in an amoeba, was delayed by EGTA, because the percentage of four-nucleate amoebae was higher than in controls at day 3 of incubation. EDTA made metacystic development unusual by producing a large number of metacystic amoebae with more than ten nuclei. The inhibition of excystation by these chelators was partially abrogated by their removal. A putative antagonist of intracellular calcium flux, 8-( N,N-diethylamino) octyl-3,4,5-trimethoxybenzoate (TMB-8) also inhibited the excystation and metacystic development, but had little effect on cyst viability. The slow Na(+)-Ca(2+) channel blocker bepridil but not verapamil inhibited the excystation and metacystic development, associating with reduced cyst viability at higher concentrations. The inhibitory effect of bepridil on excystation was abrogated by removal of the drug. The CaM inhibitor trifIuoperazine (TFP) but not W-7 [ N-(6-aminohexyl)-chloro-1-naphtalene sulphonamide] inhibited the excystation and metacystic development. The inhibitory effect of TFP on excystation was also abrogated by removal of the drug. These results indicate that extracellular calcium ions, amoebic intracellular calcium flux, calcium channels, and a CaM-dependent process contribute to the excystation and metacystic development of E. invadens.

Animals↗
PubMed2002

Effect of proteasome inhibitors on the growth, encystation, and excystation of Entamoeba histolytica and Entamoeba invadens.

The effect of three proteasome inhibitors, lactacystin, clasto-lactacystin beta-lactone, and MG-132, on the growth, encystation, and excystation of Entamoeba histolytica and Entamoeba invadens was examined. All of these drugs blocked E. histolytica growth in a concentration-dependent manner; lactacystin was most potent for the inhibition and MG-132 showed the inhibitory effect only at higher concentrations. E. invadens was more resistant to these drugs than E. histolytica. Encystation of E. invadens was also inhibited and was more sensitive to the drugs than was growth. Beta-lactone was the most potent encystation inhibitor. The inhibitory effect of lactacystin and the beta-lactone on encystation was slightly and little abrogated by the removal of the drug, respectively. Multinucleation occurred in E. histolytica trophozoites treated with these drugs, being most marked with lactacystin. In contrast, no multinucleation was observed in E. invadens treated with the drugs. Electron microscopy revealed that the treatment of E. histolytica trophozoites with lactacystin led to an increase in the number of cells with many glycogen granules in the cytoplasm. Lactacystin, beta-lactone and MG-132 had no or little effect on the excystation and metacystic development of E. invadens. These results suggest that proteasome function plays an important role for Entamoeba growth and encystation, but has no obvious effect on excystation or metacystic development.

Animals↗
PubMed2002

Inhibition of excystation and metacystic development of Entamoeba invadens by the dinitroaniline herbicide oryzalin.

The effect of oryzalin on excystation and metacystic development of Entamoeba invadens strain IP-1 was examined by transfer of cysts to a growth medium containing the drug. Excystation, which was assessed by counting the number of metacystic amoebae after induction of excystation, was inhibited by oryzalin in a concentration-dependent manner. Metacystic development, which was determined by the number of nuclei in metacystic amoebae, was also inhibited by oryzalin because the percentage of 4-nucleate amoebae at day 1 remained unchanged at day 3. The addition of oryzalin after the induction of excystation decreased the number of metacystic amoebae, compared with control cultures. When cysts were incubated for 1 day in growth medium plus oryzalin, little increase in the number of metacystic amoebae was observed after removal of the drug. Excystation and metacystic development were further inhibited when the cysts were incubated for 30 min in encystation medium containing oryzalin before transfer to growth medium with the drug. When cysts were incubated for 30 min in encystation medium before transfer to growth medium without the drug, metacystic amoebae decreased in number. Pretreatment of cysts with oryzalin for 30 min in phosphate-buffered saline markedly reduced viability and prevented excystation in growth medium with or without the drug. The results indicate that oryzalin inhibits excystation and metacystic development of E. invadens, suggesting that it may be an inhibitor of Entamoeba infection.

Animals↗
PubMedDate not supplied

Entamoeba invadens: inhibition of excystation and metacystic development by aphidicolin.

The effect of aphidicolin, a specific inhibitor of the replicative DNA polymerases, on the excystation and metacystic development of Entamoeba invadens was examined. The protein profile of metacystic amoebae and their immunogenicity in the presence and absence of aphidicolin were also examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. Excystation, which was assessed by counting the number of metacystic amoebae after the induction of excystation, was inhibited by aphidicolin in a concentration-dependent manner during incubation compared to the controls. Metacystic development, when determined by the number of nuclei in amoeba, was also inhibited by aphidicolin, because the percentage of 4-nucleate amoebae in cultures with aphidicolin during incubation was higher than that in cultures without the drug. The addition of aphidicolin to cultures at day 1 of incubation reduced the number of metacystic amoebae thereafter compared to cultures without the drug. The inhibitory effect of aphidicolin on excystation and metacystic development was reversed by removal of the drug. Pretreatment of cysts with aphidicolin before transfer to a growth medium containing the drug had no further effect on the excystation and metacystic development. Cellular proteins of metacystic amoebae with 4 nuclei, which were predominant even at day 3 in the cultures with aphidicolin, reacted strongly with rabbit anticyst serum absorbed with trophozoite proteins. In contrast, those of metacystic amoebae with 1 nucleus, which were predominant at day 3 in cultures without aphidicolin, no longer reacted with the absorbed anticyst serum, suggesting change in the expression of proteins during metacystic development.

Animals↗