• Journal of Microbiology and Biotechnology
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• v.29 no.5
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• pp.713-720
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• 2019

Acanthamoeba castellanii belonging to the T4 genotype may cause a fatal brain infection known as granulomatous amoebic encephalitis, and the vision-threatening eye infection Acanthamoeba keratitis. The aim of this study was to evaluate the antiamoebic effects of three clinically available antidiabetic drugs, Glimepiride, Vildagliptin and Repaglinide, against A. castellanii belonging to the T4 genotype. Furthermore, we attempted to conjugate these drugs with silver nanoparticles (AgNPs) to enhance their antiamoebic effects. Amoebicidal, encystation, excystation, and host cell cytotoxicity assays were performed to unravel any antiacanthamoebic effects. Vildagliptin conjugated silver nanoparticles (Vgt-AgNPs) characterized by spectroscopic techniques and atomic force microscopy were synthesized. All three drugs showed antiamoebic effects against A. castellanii and significantly blocked the encystation. These drugs also showed significant cysticidal effects and reduced host cell cytotoxicity caused by A. castellanii. Moreover, Vildagliptin-coated silver nanoparticles were successfully synthesized and are shown to enhance its antiacanthamoebic potency at significantly reduced concentration. The repurposed application of the tested antidiabetic drugs and their nanoparticles against free-living amoeba such as Acanthamoeba castellanii described here is a novel outcome that holds tremendous potential for future applications against devastating infection.

• Parasites, Hosts and Diseases
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• v.61 no.4
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• pp.388-396
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• 2023

Entamoeba histolytica is an enteric tissue-invasive protozoan parasite causing amoebic colitis and liver abscesses in humans. Amoebic contact with host cells activates intracellular signaling pathways that lead to host cell death via generation of caspase-3, calpain, Ca²⁺ elevation, and reactive oxygen species (ROS). We previously reported that various NADPH oxidases (NOXs) are responsible for ROS-dependent death of various host cells induced by amoeba. In the present study, we investigated the specific NOX isoform involved in ROS-dependent death of hepatocytes induced by amoebas. Co-incubation of hepatoma HepG2 cells with live amoebic trophozoites resulted in remarkably increased DNA fragmentation compared to cells incubated with medium alone. HepG2 cells that adhered to amoebic trophozoites showed strong dichlorodihydrofluorescein diacetate (DCF-DA) fluorescence, suggesting intracellular ROS accumulation within host cells stimulated by amoebic trophozoites. Pretreatment of HepG2 cells with the general NOX inhibitor DPI or NOX2-specific inhibitor GSK 2795039 reduced Entamoeba-induced ROS generation. Similarly, Entamoeba-induced LDH release from HepG2 cells was effectively inhibited by pretreatment with DPI or GSK 2795039. In NOX2-silenced HepG2 cells, Entamoeba-induced LDH release was also significantly inhibited compared with controls. Taken together, the results support an important role of NOX2-derived ROS in hepatocyte death induced by E. histolytica.

• Animal cells and systems
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• v.6 no.3
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• pp.227-232
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• 2002

GroESLx proteins of symbiotic X-bacteria were overproduced in Escherichia coli and their structural characteristics were assayed after simple purification. The GroESx and GroELx were heat-stable at 8$0^{\circ}C$ and 5$0^{\circ}C$, respectively. After heat-treatment, GroESx was purified by DEAE Sephadex A-50 chromatography and GroELx was purified by step- and linear sucrose density gradient ultracentrifugation. Molecular masses of GroESx and GroELx were 50-80 kDa and 800 kDa, respectively, as estimated by sucrose density gradient ultracentrifugation. In chemical cross-linking analysis, subunits of GroESx were mostly cross-linked by incubation for 3 h in 0.4% glutaralde-hyde and GroESx was found to be composed of homo-heptamer subunits. Those of GroELx were cross-linked within 10 min in 0.3% glutaraldehyde and GroELx was in two stacks of homo-heptamer subunits. On the other hand, GroESx and GroELx proteins in a solution could not be cross-linked even after incubation for 3 h in 0.5% glutaraldehyde. GroELx was stable at 4-37$^{\circ}C$. In the presence of both GroESx and ATP, GroELx$_{14}$ was stable at 37$^{\circ}C$ but not at 4$^{\circ}C$ or 24$^{\circ}C$. Thus, we confirmed the oligomeric properties of GroESx$_{7}$ and GroELx$_{14}$ and their stability to heat and in the interaction with GroESx.x.

• Applied Microscopy
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• v.30 no.4
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• pp.403-412
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• 2000

Urechis unicinctus spermatogenic cells, sperm and spermatids, prepared from testis are investigated to identify nuclear actin using amoeba monorlonal anti-actin as the first Ab and gold particles (10 nm) conjugated mouse IgG (immunogold) as the Ab marker. The Ag-Ab reactions analyzed the localization of nuclear actin of the spermatogenic cells and the immunogold particles incorporated mainly with nuclear matrices. A few immunogold particles are merged into the acrosomes and the other architectures of spermatogenic cells, such as mitochondrion and centrioles. It is often observed and there is a tendency in which the incorporated immunogold particles are increased in number in the nuclear matrices of sperm compared with that of spermatids The increments and decrements of the incorporated immunogold particles according to developmental stages and the spermatogenic architec-tures are interpreted and discussed in aspect of acrosomal function and of nuclear condensation of spermatids.

• Parasites, Hosts and Diseases
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• v.44 no.4 s.140
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• pp.331-341
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• 2006

Actin binding proteins play key roles in cell structure and movement particularly as regulators of the assembly, stability and localization of actin filaments in the cytoplasm. In the present study, a cDNA clone encoding an actin bundling protein named as AhABP was isolated from Acanthamoeba healyi, a causative agent of granulomatous amebic encephalitis. This clone exhibited high similarity with genes of Physarum polycephalum and Dictyostelium discoideum, which encode actin bundling proteins. Domain search analysis revealed the presence of essential conserved regions, i.e., an active actin binding site and 2 putative calcium binding EF-hands. Transfected amoeba cells demonstrated that AhABP is primarily localized in phagocytic cups, peripheral edges, pseudopods, and in cortical cytoplasm where actins are most abundant. Moreover, AhABP after the deletion of essential regions formed ellipsoidal inclusions within transfected cells. High-speed co-sedimentation assays revealed that AhABP directly interacted with actin in the presence of up to $10{\mu}M$ of calcium. Under the electron microscope, thick parallel bundles were formed by full length AhABP, in contrast to the thin actin bundles formed by constructs with deletion sites. In the light of these results, we conclude that AhABP is a novel actin bundling protein that is importantly associated with actin filaments in the cytoplasm.

• Parasites, Hosts and Diseases
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• v.40 no.1
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• pp.17-24
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• 2002

We have cloned a cDNA encoding a cysteine proteinase of the Acanthamoeba healui OC-3A strain isolated from the brain of a granulomatous amoebic encephalitis patient. A DNA probe for an A. healui cDNA library screening was amplified by PCR using degenerate oligonucleotide primers designed on the basis of conserved amino acids franking the active sites of cysteine and asparagine residues that are conserved in the eukaryotic cysteine proteinases. Cysteine proteinase gene of A. healui (AhCPI) was composed of 330 amino acids with signal sequence, a proposed pro-domain and a predicted active site made up of the catalytic residues, $Cys^{25},{\;}His^{159},{\;}and{\;}Asn^{175}$. Deduced amino acid sequence analysis indicates that AhCPI belong to ERFNIN subfamily of C 1 peptidases. By Northern blot analysis. no direct correlation was observed between AhCPI mRNA expression and virulence of Acanthamoeba, but the gene was expressed at higher level in amoebae isolated from soil than amoeba from clinical samples. These findings raise the possibility that AhCPI protein may play a role in protein metabolism and digestion of phagocytosed bacteria or host tissue debris rather than in invasion of amoebae into host tissue.

• Parasites, Hosts and Diseases
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• v.55 no.3
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• pp.233-238
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• 2017

Pathogenic Naegleria fowleri, Acanthamoeba castellanii, and Acanthamoeba polyphaga, are distributed worldwide. They are causative agents of primary amoebic meningoencephalitis or acanthamoebic keratitis in humans, respectively. Trophozoites encyst in unfavorable environments, such as exhausted food supply and desiccation. Until recently, the method of N. fowleri encystation used solid non-nutrient agar medium supplemented with heat-inactivated Escherichia coli; however, for the amoebic encystment of Acanthamoeba spp., a defined, slightly modified liquid media is used. In this study, in order to generate pure N. fowleri cysts, a liquid encystment medium (buffer 1) modified from Page's amoeba saline was applied for encystation of N. fowleri. N. fowleri cysts were well induced after 24 hr with the above defined liquid encystment medium (buffer 1). This was confirmed by observation of a high expression of differential mRNA of nfa1 and actin genes in trophozoites. Thus, this liquid medium can replace the earlier non-nutrient agar medium for obtaining pure N. fowleri cysts. In addition, for cyst formation of Acanthamoeba spp., buffer 2 (adjusted to pH 9.0) was the more efficient medium. To summarize, these liquid encystment media may be useful for further studies which require axenic and pure amoebic cysts.

• Parasites, Hosts and Diseases
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• v.35 no.2
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• pp.127-134
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• 1997

Transmission electron microscopy of an ArGnthnmoebo isolate (KA/LS) from a contact lens case revealed bacterial endosymbionts within cytoplasm of the amoebae. The Acnnthamoebn isolate belonged to the morphological group ll. Based on the polymerase chain reaction (PCR) - restriction fragment leilgth polymorphism (RFLP) of 185 ribosomal RNA coding DNA (rDNA) , the isolate was identified as A. Iwnunensis. Strain typing by isoenzyme analysis using isorlectric focusing (IEF) and mitochondrial (Ent) DNA RFLP revealed that the isolate was closely related with KA/Ll , the most predominant type of isolates from contact lens storage casas, KA/E2, a clinical isolate, KA/W4, previou:fly reported to host endosymbionts. and L3a strains of A. Iwnunensis. The endosymbionts were similar to those of KA/W4 in a.jpects that they were randomly distributed in both trophozoites and cysts, and were rod-shaped bacteri3 measuring approximately 1.38 x 0.50 ㎛. But the number of endosymbionts per amoeba was significantly lower than that of KA/W4. They were neither limited by phagosomal membranes nor included in lacunae- like stnlcture.

• Parasites, Hosts and Diseases
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• v.47 no.2
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• pp.103-107
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• 2009

The genus Acanthamoeba can cause severe infections such as granulomatous amebic encephalitis and amebic keratitis in humans. However, little genomic information of Acanthamoeba has been reported. Here, we constructed Acanthamoeba expressed sequence tags (EST) database (Acanthamoeba EST DB) derived from our 4 kinds of Acanthamoeba cDNA library. The Acanthamoeba EST DB contains 3,897 EST generated from amebae under various conditions of long term in vitro culture, mouse brain passage, or encystation, and downloaded data of Acanthamoeba from National Center for Biotechnology Information (NCBI) and Taxonomically Broad EST Database (TBestDB). The almost reported eDNA/genomic sequences of Acanthamoeba provide stand alone BLAST system with nucleotide (BLAST NT) and amino acid (BLAST AA) sequence database. In BLAST results, each gene links for the significant information including sequence data, gene orthology annotations, relevant references, and a BlastX result. This is the first attempt for construction of Acanthamoeba database with genes expressed in diverse conditions. These data were integrated into a database (http://www. amoeba.or.kr).

• Journal of agricultural medicine and community health
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• v.6 no.1
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• pp.68-72
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• 1981

This study was undertaken to evaluate the status of intestinal protozoan infections in the children of specific groups. A total number of 441(male 250; female 191) consisting of 224 children of national feeble minded children institute, 59 Han-Bit blindness students, 158 children of local mental retardation children institute(Kang-Dong Ku) of specific groups were examined. The method employed was formalin-ether sedimentation technique with Lugol's iodine stain. The results were as follows: 1) The positive rates for any kinds of protozoan cysts were 13.8%(72 positives out of 441 samples) with sexual difference of 14.0% in male and 12.6% in female. 2) In the institute differences of positive rates of protozoan cysts were 15.2% in national feeble minded children institute, 16.9% in Han-Bit blindness school, and local mental retardation children institute(Kang-Dong Ku). 3) Cysts of Entamoeba coli were recovered in 8.6% and cysts of pathogenic amoeba, Entamoeba histolytica were found in 1.4%. Cysts of Endolimax nana (3.4%). Giardia lamblia (2.9%) were found in this study. 4) The degree of multiple infections of protozoan cysts were; single infection 81.4%, double infection 16.9% and quadriple infection 1.7% respectively.