A survey was carried out from August to December 2004 in Pusan, Korea to document the presence of free-living amoeba (FLA), including the genus Acanthamoeba, in both contact lens storage cases and domestic tap water. Acanthamoeba was isolated from 5 (4.2%) in 120 contact lens storage cases. Four house tap water samples from residents, whose contact lens storage cases had been contaminated by Acanthamoeba, were also found to be contaminated with Acanthamoeba. Therefore, the contamination rate of FLA and Acanthamoeba in domestic tap water was investigated in order to examine the role of domestic tap water in Acanthamoeba contamination of contact lens storage cases. FLA and Acanthamoeba were identified in 97 (46.8%) and 16 (7.7%) of the 207 domestic tap water samples, respectively. There were no significant differences between the contamination rates of FLA in tap water according to the filtration plant of origin. No FLA was detected in the tap water directly supplied by the water purification plants. Water storage tanks appear to promote FLA colonization, including Acanthamoeba, in domestic tap water. This increases the risk of Acanthamoeba contamination in contact lens storage cases as well as increasing the risk of Acanthamoeba keratitis.
Acanthamoeba/*isolation & purification ; Amebiasis/epidemiology ; Animals ; Comparative Study ; Contact Lenses/parasitology ; Data Collection ; Humans ; Korea/epidemiology ; Research Support, Non-U.S. Gov't ; Risk Factors ; Water/*parasitology ; Water Supply/*standards

The cyclophilins (Cyps) are family members of proteins that exhibit peptidylprolyl cis-trans isomerase (PPIase, EC 5.2.1.8) activity and bind the immunosuppressive agent cyclosprin A (CsA) in varying degrees. During the process of random sequencing of a cDNA library made from Giardia intestinalis WB strain, the cyclophilin gene (gicyp 1) was isolated. An open reading frame of gicyp 1 gene was 576 nucleotides, which corresponded to a translation product of 176 amino acids (Gicyp 1). The identity with other Cyps was about 58-71%. The 13 residues that constituted the CsA binding site of human cyclophilin were also detected in the amino acid sequence of Gicyp 1, including tryptophan residue essential for the drug binding. The single copy of the gicyp 1 gene was detected in the G. intestinalis chromosome by southern hybridization analysis. Recombinant Gicyp 1 protein clearly accelerated the rate of cis-- Amino Acid Sequence ; Animals ; Cloning, Molecular ; Cyclophilins/antagonists & inhibitors/chemistry/genetics/*isolation&purification ; Cyclosporine/metabolism/pharmacology ; Giardia lamblia/*chemistry/genetics ; Human ; Immunosuppressive Agents ; Molecular Sequence Data ; Protein Binding ; Protozoan Proteins ; Recombinant Proteins

In order to evaluate the possible roles of secretory proteases in the pathogenesis of amoebic keratitis, we purified and characterized a serine protease secreted by Acanthamoeba lugdunensis KA/E2, isolated from a Korean keratitis patient. The ammonium sulfate-precipitated culture supernatant of the isolate was purified by sequential chromatography on CM-Sepharose, Sephacryl S-200, and mono Q-anion exchange column. The purified 33 kDa protease had a pH optimum of 8.5 and a temperature optimum of 55 degrees C. Phenylmethylsulfonylfluoride and 4- (2- Aminoethyl) -benzenesulfonyl-fluoride, both serine protease specific inhibitors, inhibited almost completely the activity of the 33 kDa protease whereas other classes of inhibitors did not affect its activity. The 33 kDa enzyme degraded various extracellular matrix proteins and serum proteins. Our results strongly suggest that the 33 kDa serine protease secreted from this keratopathogenic Acanthamoeba play important roles in the pathogenesis of amoebic keratitis, such as in corneal tissue invasion, immune evasion and nutrient uptake.
Acanthamoeba/*enzymology/isolation & purification/pathogenicity ; Acanthamoeba Keratitis/*parasitology ; Animals ; Cornea/parasitology ; Humans ; Hydrogen-Ion Concentration ; Korea ; Serine Endopeptidases/chemistry/*isolation & purification/*metabolism ; Substrate Specificity ; Temperature ; Virulence Factors

Four hundred and sixty five randomly selected clones from a cDNA library of Blattella germanica were partially sequenced and searched using BLAST as a means of analyzing the transcribed sequences of its genome. A total of 363 expressed sequence tags (ESTs) were generated from 465 clones after editing and trimming the vector and ambiguous sequences. About 42% (154/363) of these clones showed significant homology with other data base registered genes. These new B. germanica genes constituted a broad range of transcripts distributed among ribosomal proteins, energy metabolism, allergens, proteases, protease inhibitors, enzymes, translation, cell signaling pathways, and proteins of unknown function. Eighty clones were not well-matched by database searches, and these represent new B. germanica-specific ESTs. Some genes which drew our attention are discussed. The information obtained increases our understanding of the B. germanica genome.
Sequence Alignment ; Reverse Transcriptase Polymerase Chain Reaction ; Molecular Sequence Data ; Male ; Female ; *Expressed Sequence Tags ; Blattellidae/*genetics ; Base Sequence ; Animals

The taxonomy of Acanthamoeba spp., an amphizoic amoeba which causes granulomatous amoebic encephalitis and chronic amoebic keratitis, has been revised many times. The taxonomic validity of some species has yet to be assessed. In this paper, we analyzed the morphological characteristics, nuclear 18s rDNA and mitochondrial 16s rDNA sequences and the Mt DNA RFLP of the type strains of four Acanthamoeba species, which had been previously designated as A. divionensis, A. parasidionensis, A. mauritaniensis, and A. rhysodes. The four isolates revealed characteristic group II morphology. They exhibited 18S rDNA sequence differences of 0.2-1.1% with each other, but more than 2% difference from the other compared reference strains. Four isolates formed a different clade from that of A. castellanii Castellani and the other strains in morphological group II on the phylogenetic tree. In light of these results, A. paradivionensis, A. divionensis, and A. mauritaniensis should be regarded as synonyms for A. rhysodes.
Acanthamoeba/*classification/*genetics ; Animals ; DNA, Mitochondrial/genetics ; DNA, Ribosomal/genetics ; Phylogeny ; Polymorphism, Restriction Fragment Length ; RNA, Ribosomal, 18S/genetics

BACKGROUND: This study aimed to evaluate the adhesion of Acanthamoeba trophozoites on cosmetic contact lenses (CLs) with and without CL care multipurpose solution (MPS) treatment. METHODS: Acanthamoeba lugdunensis L3a trophozoites were inoculated onto disks trimmed from CLs: 1-day Acuvue moist, 1-day Acuvue define, Acuvue 2, and Acuvue 2 define. After 18-hour inoculation, the number of adherent trophozoites was counted under phase contrast microscopy. The effects of MPS, Opti-Free Express, soaking CLs for 6 hours, on Acanthamoeba adhesion were analyzed. Scanning electron microscopic examination was performed for assessment of Acanthamoeba attached on the lens surface. RESULTS: Acanthamoeba trophozoites showed greater adhesion to cosmetic CL (P = 0.017 for 1-day CL and P = 0.009 for 2-week CL) although there was no significant difference between the types of cosmetic CL. On all lenses, the number of adherent Acanthamoeba was significantly reduced after treatment with MPS (P < 0.001 for 1-day Acuvue moist, P = 0.046 for 1-day Acuvue define, P < 0.001 for Acuvue 2, and P = 0.015 for Acuvue 2 define), but there was still significant difference between conventional and cosmetic CLs (P = 0.003 for 1-day CL and P < 0.001 for 2-week CL, respectively). More attachment of Acanthamoeba was observed on colored area and the acanthopodia of Acanthamoeba was placed on the rough surface of colored area. CONCLUSION: Acanthamoeba showed a greater affinity for cosmetic CL and mostly attached on colored area. Although MPS that contained myristamidopropyl dimethylamine reduced the adhesion rate, there was a significant difference between conventional and cosmetic CLs.
Acanthamoeba ; Contact Lenses ; Microscopy, Phase-Contrast ; Trophozoites

Although helminth parasites have different life cycles, their hosts share similar immune responses involving Th2 cell-type. Here, we extracted proteins from the larvae of Anisakis simplex complex and Trichinella spiralis to identify common and specific antigens (or allergens) associated with the Th2 immune response. We performed two-dimensional electrophoresis analysis and Matrix-assisted laser desorption ionization–time of flight/time of flight (MALDI-TOF/TOF) experiments. We found 13 potentially immunogenic proteins, which included 5 spots specific to T. spiralis and 8 common to T. spiralis and A. simplex, by tandem mass spectrometry. These molecules were identified structurally as actin, tropomyosin, col cuticle N domain-containing protein, and heat shock proteins. We also identified molecules related to parasite-host immune modulation and interactions. Our results may contribute to reveal potential roles of immunological proteins in parasite-derived immune modulation.

Although helminth parasites have different life cycles, their hosts share similar immune responses involving Th2 cell-type. Here, we extracted proteins from the larvae of Anisakis simplex complex and Trichinella spiralis to identify common and specific antigens (or allergens) associated with the Th2 immune response. We performed two-dimensional electrophoresis analysis and Matrix-assisted laser desorption ionization–time of flight/time of flight (MALDI-TOF/TOF) experiments. We found 13 potentially immunogenic proteins, which included 5 spots specific to T. spiralis and 8 common to T. spiralis and A. simplex, by tandem mass spectrometry. These molecules were identified structurally as actin, tropomyosin, col cuticle N domain-containing protein, and heat shock proteins. We also identified molecules related to parasite-host immune modulation and interactions. Our results may contribute to reveal potential roles of immunological proteins in parasite-derived immune modulation.

Although helminth parasites have different life cycles, their hosts share similar immune responses involving Th2 cell-type. Here, we extracted proteins from the larvae of Anisakis simplex complex and Trichinella spiralis to identify common and specific antigens (or allergens) associated with the Th2 immune response. We performed two-dimensional electrophoresis analysis and Matrix-assisted laser desorption ionization–time of flight/time of flight (MALDI-TOF/TOF) experiments. We found 13 potentially immunogenic proteins, which included 5 spots specific to T. spiralis and 8 common to T. spiralis and A. simplex, by tandem mass spectrometry. These molecules were identified structurally as actin, tropomyosin, col cuticle N domain-containing protein, and heat shock proteins. We also identified molecules related to parasite-host immune modulation and interactions. Our results may contribute to reveal potential roles of immunological proteins in parasite-derived immune modulation.

Although helminth parasites have different life cycles, their hosts share similar immune responses involving Th2 cell-type. Here, we extracted proteins from the larvae of Anisakis simplex complex and Trichinella spiralis to identify common and specific antigens (or allergens) associated with the Th2 immune response. We performed two-dimensional electrophoresis analysis and Matrix-assisted laser desorption ionization–time of flight/time of flight (MALDI-TOF/TOF) experiments. We found 13 potentially immunogenic proteins, which included 5 spots specific to T. spiralis and 8 common to T. spiralis and A. simplex, by tandem mass spectrometry. These molecules were identified structurally as actin, tropomyosin, col cuticle N domain-containing protein, and heat shock proteins. We also identified molecules related to parasite-host immune modulation and interactions. Our results may contribute to reveal potential roles of immunological proteins in parasite-derived immune modulation.