OBJECTIVETo identify the binding site on glycophorin A (GPA) for EBA-175 to provide clue for developing short peptide vaccine and therapeutic agents against Plasmodium falciparum.

METHODSWith the recombinant protein of EBA-175 as the target molecule, the mimetic peptides of GPA were screened from a 12-mer random peptide library. Three rounds of biopanning were carried out, and enzyme-linked immunosorbent assay (ELISA), competitive ELISA, Dot-ELISA and Western blotting used to evaluate the binding between the phage-borne peptides and EBA-175. The insert DNA sequences of positive clones were determined and their amino acid sequences deduced.

RESULTSThirty clones from the third round were randomly selected, of which 27 were found positive by sandwich ELISA. Competitive ELISA proved that most of the phage-borne peptides could competitively inhibit the binding of antibody (EBA-175 Ab) with EBA-175. Analysis of DNA and amino acid sequences indicated that 24 positive phage clones contained the conservative sequence of IRR, which was highly homologous with the 114-116 amino acids of GPA.

CONCLUSIONThese phage-displayed peptides can bind with EBA-175, and the amino acid sequence IRR might play an important role in the binding between EBA-175 and GPA.

Antigens, Protozoan ; metabolism ; Binding Sites ; Enzyme-Linked Immunosorbent Assay ; Glycophorin ; chemistry ; Humans ; Peptide Library ; Plasmodium falciparum ; Protozoan Proteins ; metabolism ; Sequence Analysis, DNA ; Sequence Analysis, Protein

Randomly selected 435 clones from Acanthamoeba healyi cDNA library were sequenced and a total of 387 expressed sequence tags (ESTs) had been generated. Based on the results of BLAST search, 130 clones (34.4%) were identified as the genes encoding surface proteins, enzymes for DNA, energy production or other metabolism, kinases and phosphatases, protease, proteins for signal transduction, structural and cytoskeletal proteins, cell cycle related proteins, transcription factors, transcription and translational machineries, and transporter proteins. Most of the genes (88.5%) are newly identified in the genus Acanthamoeba. Although 15 clones matched the genes of Acanthamoeba located in the public databases, twelve clones were actin gene which was the most frequently expressed gene in this study. These ESTs of Acanthamoeba would give valuable information to study the organism as a model system for biological investigations such as cytoskeleton or cell movement, signal transduction, transcriptional and translational regulations. These results would also provide clues to elucidate factors for pathogenesis in human granulomatous amoebic encephalitis or keratitis by Acanthamoeba.
Acanthamoeba/cytology/*genetics/pathogenicity ; Amebiasis/parasitology ; Animals ; DNA, Protozoan/*genetics ; *Expressed Sequence Tags ; Gene Library ; Human ; Protozoan Proteins/genetics ; *Sequence Analysis, DNA ; Signal Transduction ; Support, Non-U.S. Gov't

Piroplasms are tick-transmitted, intracellular, hemoprotozoan parasites that cause anorexia, fever, anemia, and icterus. Theileriosis is caused by Theileria sergenti and causes major economic losses in grazing cattle in Japan and Korea. In May 2003, we examined the antigenic diversity of the major piroplasm surface protein (MPSP) gene in 35 healthy Jeju black cattle that were born and raised at the National Institute of Subtropical Agriculture. On microscopic examination of Giemsa-stained blood smears, 9 of 35 cattle had intra-erythrocytic piroplasms. Hematological data were within normal range for all 35 cattle. Amplification of DNA from all blood samples using universal MPSP gene primers showed mixed infections with C, I, and B type Theileria spp. Type C was identified in 20 of 35 blood samples, and type B was identified in 17 samples. Allelic variation was seen in type B.
Animals ; Antigens, Protozoan/*genetics ; Base Sequence ; Cattle ; DNA Primers/genetics ; *Genetic Variation ; Korea ; Molecular Sequence Data ; *Phylogeny ; Protozoan Proteins/*genetics ; Sequence Analysis, DNA ; Theileria/*genetics ; Theileriasis/*parasitology

An ELISA was established to measure bovine IgG directed against the recombinant antigenic determinant of Nc-p43, a major surface antigen of Neospora caninum. In a previous study, two thirds of the Cterminal of the molecule was expressed as a 6 x His tagged protein (Ncp43P) for ELISA using 2/3 of the N-terminal of SAG1 from Toxoplasma gondii as a control (TgSAG1A). Among 852 cattle sera collected from stock farms scattered nation-wide, 103 sera (12.1%) were found to react with Ncp43P positively, but no positive reaction was observed with TgSAG1A. This study shows that Ncp43P could be available as an efficient antigen for the diagnosis of neosporosis in cattle. Furthermore, it together with TgSAG1A, could be useful for the differential diagnosis of N. caninum and T. gondii infections in other mammals.
Animals ; Antibodies, Protozoan ; Cattle ; Cattle Diseases/diagnosis/epidemiology ; Coccidiosis/diagnosis/epidemiology/veterinary ; Enzyme-Linked Immunosorbent Assay/methods/*veterinary ; Immunoglobulin G/*analysis ; Neospora/*immunology ; Protozoan Proteins/*immunology ; Seroepidemiologic Studies

Identification of expressed protein profiles and antigenic determination are some of the most challenging aspects of proteomics. Two-dimensional gel electrophoresis (2-DE) combined with immunoblot analysis were employed to study the N. caninum proteome. Protein sample preparation was carried out by first conducting sonication, followed by adding lysis buffer containing 7M urea plus 2M thiourea to the purified tachyzoites in order to complete disruption. A total of 335 differentially expressed protein spots were detected using pH 4-7 IPG strip (7 cm) that were run in a 56 kVh isoelectric focusing (IEF) system. Of the spots analyzed, 64 were identified as antigenic spots on immunoblot profile. Major antigenic spots appeared at 65 kDa (pI 5.2-5.3), 51 kDa (pI 5.5), 38 kDa (pI 5.1), 33 kDa (pI 4.4), 29 kDa (pI 5.6) and 15.5 kDa (pI 5.0) were observed to be significantly distinct compared to the rest of the antigenic spots. The results indicate that combination of 2-DE and immunoblotting methods were thought as very useful tools in defining both proteins and antigens of N. caninum tachyzoites. Additionally, present 2-DE profiles may be valuable in further proteomic approaches and study of the pathogen.
Animals ; Antibody Formation ; Antigens, Protozoan/*analysis/isolation&purification ; Electrophoresis, Gel, Two-Dimensional/methods ; Electrophoresis, Polyacrylamide Gel ; Epitopes/analysis ; Image Processing, Computer-Assisted ; Immunoblotting/methods ; Isoelectric Focusing ; Neospora/*chemistry/immunology ; Proteome/analysis ; Proteomics ; Protozoan Proteins/*analysis/isolation&purification

Protozoan ciliates are a group of unicellular eukaryotes. The special characteristics of stop codons usage in termination of protein biosynthesis in ciliates cells makes them an ideal model to study the mechanism of stop codon recognition of polypeptides release factors. To localize the functional positions of biomolecules in ciliates cell, we constructed a macronuclear artificial chromosome containing a gene encoding red fluorescence protein (EoMAC_R) based on the structural characteristics of ciliates chromosome. Three factors, L11, eRF1a, and eRF3 that are involved in termination process of protein synthesis were colocalized in Euplotes octocarinatus cells by using novel EoMAC_R and the previously constructed EoMAC_G. The results indicated that protein synthesis mainly occurred inside the "C" shape macronucleus, suggesting that EoMAC could be a useful tool for localizing biomolecules in ciliates cell.
Chromosomes, Artificial ; Codon, Terminator ; metabolism ; Euplotes ; chemistry ; Peptide Termination Factors ; analysis ; genetics ; metabolism ; Peptides ; metabolism ; Protein Biosynthesis ; genetics ; Protozoan Proteins ; analysis ; genetics ; Ribosomal Proteins ; analysis ; genetics

OBJECTIVETo construct a recombinant prokaryotic expression vector (plasmid) containing microneme protein 1 (MIC1) partial gene in toxoplasma gondii (T. gondii) ZS2 isolate. The gene was expressed in varied Escherichia coli (E. coli) after sequencing.

METHODSThe gene fragment coding MIC 1 from the genomic DNA of T. gondii ZS2 isolate was amplified by polymerase chain reaction (PCR). The gene was inserted to a prokaryotic expression vector pWR450-1 by digesting with restriction enzymes and linking reaction. The positive clone was screened on LB plates containing ampicillin and identified by restrictive enzyme digestion, PCR amplification and sequence analysis. The recombinant plasmid was transferred into E. coli TG1, JM109 (DE3) and DH5 alpha, and was expressed under the induction of IPTG. The expression products were identified by SDS-PAGE. The MIC1 gene structure was analyzed and compared in homology with the gene sequence of RH isolate using computer software.

RESULTSThe recombinant plasmid pWR450-1/MIC1, after cloning from acquired 471 bp MIC1 gene fragment and amplified from the genome gene ZS2, was complete homologous to the sequence of RH isolate, reflecting its highly conservative. The gene could be expressed as fusion protein with 70,000 in varied E. coli.

CONCLUSIONRecombinant plasmid pWR450-MIC1 was successfully constructed and could be expressed in different strains of E. coli, laying a foundation for research on its structure and function.

Animals ; Base Sequence ; Cell Adhesion Molecules ; biosynthesis ; genetics ; Cloning, Molecular ; DNA, Protozoan ; analysis ; Escherichia coli ; genetics ; Gene Expression ; Molecular Sequence Data ; Nucleic Acid Amplification Techniques ; Protozoan Proteins ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Toxoplasma ; genetics

Acanthamoeba infection is difficult to treat because of the resistance property of Acanthamoeba cyst against the host immune system, diverse antibiotics, and therapeutic agents. To identify encystation mediating factors of Acanthamoeba, we compared the transcription profile between cysts and trophozoites using microarray analysis. The DNA chip was composed of 12,544 genes based on expressed sequence tag (EST) from an Acanthamoeba ESTs database (DB) constructed in our laboratory, genetic information of Acanthamoeba from TBest DB, and all of Acanthamoeba related genes registered in the NCBI. Microarray analysis indicated that 701 genes showed higher expression than 2 folds in cysts than in trophozoites, and 859 genes were less expressed in cysts than in trophozoites. The results of real-time PCR analysis of randomly selected 9 genes of which expression was increased during cyst formation were coincided well with the microarray results. Eukaryotic orthologous groups (KOG) analysis showed an increment in T article (signal transduction mechanisms) and O article (posttranslational modification, protein turnover, and chaperones) whereas significant decrement of C article (energy production and conversion) during cyst formation. Especially, cystein proteinases showed high expression changes (282 folds) with significant increases in real-time PCR, suggesting a pivotal role of this proteinase in the cyst formation of Acanthamoeba. The present study provides important clues for the identification and characterization of encystation mediating factors of Acanthamoeba.
Acanthamoeba castellanii/*genetics/physiology ; Animals ; Cluster Analysis ; Databases, Genetic ; Expressed Sequence Tags ; Gene Expression Profiling ; Gene Expression Regulation, Developmental/*genetics ; Oligonucleotide Array Sequence Analysis ; Oocysts/*physiology ; Protozoan Proteins/*genetics ; RNA, Protozoan/genetics ; Trophozoites/*physiology

The present study was designed to investigate polymorphism in Duffy binding protein (DBP) gene of Plasmodium vivax isolates of Korea. Thirty samples were obtained from P. vivax patients in Yonchon-gun, Kyonggi-do in 1998. The PCR products of the samples were subjected to sequencing and hybridization analyses of the regions II and IV of P. vivax DBP gene. Two genotypes, SK-1 and SK-2, were identified on the basis of amino acid substitution and deletion. The genotype of 10 isolates was SK-1 and that of 20 isolates was SK-2. Most of the predicted amino acids in the region II of DBP gene were conserved between the Korean isolates and Belem strain except for 4-5 amino acid substitutions. In the region IV of DBP, a 6-bp insert that was shown in the Sal-1 allele type was found in SK-1, and a 27-bp insert that was shown in the Papua New Guinea allele type was found in SK-2. In conclusion, the present findings suggest that two genotypes of P. vivax coexist in the endemic area of Korea.
Amino Acid Sequence ; Animals ; *Antigens, Protozoan ; Base Sequence ; Carrier Proteins/*analysis/chemistry/*genetics ; DNA, Protozoan/genetics ; Genotype ; Human ; Korea ; Malaria, Vivax/parasitology ; Molecular Sequence Data ; Plasmodium vivax/*genetics/isolation & purification ; Polymerase Chain Reaction ; *Polymorphism (Genetics) ; *Protozoan Proteins ; Receptors, Cell Surface/*analysis/chemistry/*genetics ; Support, Non-U.S. Gov't

Autophagy is a catabolic process involved in the degradation of a cell's own components for cell growth, development, homeostasis, and the recycling of cellular products. Autophagosome is an essential component in the protozoan parasite during differentiation and encystation. The present study identified and characterized autophagy-related protein (Atg) 3, a member of Atg8 conjugation system, in Acanthamoeba castellanii (AcAtg3). AcAtg3 encoding a 304 amino acid protein showed high similarity with the catalytic cysteine site of other E2 like enzymes of ubiquitin system. Predicted 3D structure of AcAtg3 revealed a hammer-like shape, which is the characteristic structure of E2-like enzymes. The expression level of AcAtg3 did not increase during encystation. However, the formation of mature cysts was significantly reduced in Atg3-siRNA transfected cells in which the production of Atg8-phosphatidylethanolamine conjugate was inhibited. Fluorescent microscopic analysis revealed that dispersed AcAtg3-EGFP fusion protein gathered around autophagosomal membranes during encystation. These results provide important information for understanding autophagic machinery through the lipidation reaction mediated by Atg3 in Acanthamoeba.
Acanthamoeba castellanii/*growth & development/*metabolism ; Animals ; Gene Knockdown Techniques ; Lipid Metabolism ; Models, Molecular ; Molecular Sequence Data ; Protein Structure, Tertiary ; Protozoan Proteins/genetics ; RNA, Small Interfering/metabolism ; Rats ; Sequence Analysis, DNA ; Spores, Protozoan/*growth & development/*metabolism ; Ubiquitin-Conjugating Enzymes/genetics/*metabolism