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In Japan, amebiasis has been observed in homosexual men, in institutionalized persons, and in overseas travelers. We have previously reported an outbreak of amebiasis that occurred from 1986 to 1994 in institutions for the mentally retarded in Kanagawa and Shizuoka Prefectures in Eastern Japan. Entamoeba histolytica but not Entamoeba dispar was identified in Entamoeba cultures obtained from cyst passers in four institutions located in different municipalities in this region. In the present study, serine-rich protein genes of eight isolates from the four institutions were sequenced, and their polymorphism was analyzed. The results showed that all the sequences from the E. histolytica isolates were identical. This retrospective study led us to conclude that the outbreak of amebiasis in different municipalities was derived from a single source of E. histolytica.

Two simian Entamoeba histolytica-like strains, EHMfas1 and P19-061405, have been suggested to represent a new species based on genetic characterization. Sequence analyses of the hexokinase, glucose phosphate isomerase, and phosphoglucomutase genes supported the previous findings of isoenzyme analyses demonstrating a new zymodeme pattern. Phylogenetic studies of 18S rDNA, 5.8S rDNA, the chaperonin 60 gene, and the pyridine nucleotide transhydrogenase gene showed original clusters of simian E. histolytica-like strains below or near E. histolytica, respectively. Comparative studies of the chitinase and the serine-rich E. histolytica protein genes and locus 1-2 region revealed that most mutated units were shared among the simian E. histolytica-like strains. The similarities of each of the repeating units within the simian E. histolytica-like strains or E. histolytica and the differences of those between the both might be generated by concerted evolution. Our results indicate that EHMfas1 and P19-061405 should be considered to be the same species, despite that they were isolated from different monkey species and different habitats. Simian E. histolytica-like amebas may be endemic to macaque monkeys, as a counterpart to E. histolytica in humans, and should be differentiated from E. histolytica by the revival name Entamoeba nuttalli, as proposed for P19-061405.

fac-Re(bpy)(CO)3Cl (bpy = 2,2′-bipyridine) and similar complexes are known to act as photocatalysts for the reduction of CO2 to CO in the presence of electron donors. However, these complexes do not have significant absorption in the visible region. In a separate publication, we have reported the successful combination of fac-Re(bpy)(CO)4Cl with a zinc porphyrin that has strong absorption in the visible region to produce the ZnTMP-Re(bpy)(NHAc) dyad(5-{4-[N-4′-(rhenium(I)tricarbonylchloride-4-acetylamino-2,2′-bipyridyl)-carbamoyl]phenyl}-10,15,20-trimesityl-porphyrinatozinc(II)). In that paper we reported the photochemical reduction of CO2 using excitation of the porphyrin upper excited singlet state, S2 state (visible region), in the dyad. The very wide visible light region is able to be utilized by the sensitizer. In the present work, as regards the detailed molecular mechanism, we have demonstrated the direct electron transfer from the Zn porphyrin S2 state to the Re complex in the dyad by ultrafast tr...

Four fully human monoclonal antibodies (MAbs) toEntamoeba histolyticaintermediate subunit lectin (Igl) were prepared in XenoMouse mice, which are transgenic mice expressing human immunoglobulin loci. Examination of the reactivities of these MAbs to recombinant Igl1 and Igl2 ofE. histolyticashowed that XEhI-20 {immunoglobulin G2(κ) [IgG2(κ)]} and XEhI-28 [IgG2(κ)] were specific to Igl1, XEhI-B5 [IgG2(κ)] was specific to Igl2, and XEhI-H2 [IgM(κ)] was reactive with both Igls. Gene analyses revealed that the VHand VLgerm lines were VH3-48 and L2 for XEhI-20, VH3-21 and L2 for XEhI-28, VH3-33 and B3 for XEhI-B5, and VH4-4 and A19 for XEhI-H2, respectively. Flow cytometry analyses showed that the epitopes recognized by all of these MAbs were located on the surfaces of living trophozoites. Confocal microscopy demonstrated that most Igl1 and Igl2 proteins were colocalized on the surface and in the cytoplasm, but different localization patterns in intracellular vacuoles were also present. The preincubation of trophozoites with XEhI-20, XEhI-B5, and XEhI-H2 caused significant inhibition of the adherence of trophozoites to Chinese hamster ovary cells, whereas preincubation with XEhI-28 did not do so. XEhI-20, XEhI-B5, and XEhI-H2 were injected intraperitoneally into hamsters 24 h prior to intrahepatic challenge withE. histolyticatrophozoites. One week later, the mean abscess size in groups injected with one of the three MAbs was significantly smaller than that in controls injected with polyclonal IgG or IgM isolated from healthy humans. These results demonstrate that human MAbs to Igls may be applicable for immunoprophylaxis of amebiasis.

We recently produced human monoclonal antibody Fab fragments specific for the 19-kDa C-terminal merozoite surface protein 1 of Plasmodium falciparum in a bacterial expression system. The effect of single amino acid modifications in the third complementarity-determining regions of the heavy and light chains on affinity was examined in one of the Fab fragments, Pf25. Recombination polymerase chain reaction was used to modify Tyr(92) or Ile(97) in the light chain and Val(101) or Trp(107) in the heavy chain. No effective replacements for Tyr(92) and Val(101) were found, but possible substitutions of Ile(97) with Gly, Leu, Glu, Ala and Ser, and of Trp(107) with Arg and Ser were demonstrated. Of these modified Fab fragments, the affinities of Fabs with Ile(97)-Leu and Trp(107)-Ser mutations were slightly higher than that of the original Fab. The effects of these modifications on the antigen-antibody interaction are discussed.

SUMMARYWe have recently identified 2 surface proteins inEntamoeba histolyticaas intermediate subunits of galactose- andN-acetyl-D-galactosamine-inhibitable lectin (EhIgl1 and EhIgl2); these proteins both contain multiple CXXC motifs. Here, we report the molecular characterization of the corresponding proteins inEntamoeba dispar, which is neither pathogenic nor invasive. TwoIglgenes encoding 1110 and 1106 amino acids (EdIgl1 and EdIgl2) were cloned from 2 strains ofE. dispar. The amino acid sequence identities were 79% between EdIgl1 and EdIgl2, 75–76% between EdIgl1 and EhIgl1, and 73–74% between EdIgl2 and EhIgl2. However, all the CXXC motifs were conserved in the EdIgl proteins, suggesting that the fold conferred by this motif is important for function. Comparison of the expression level of theIglgenes by real-time RT-PCR showed 3–5 times higher expression ofEdIgl1compared toEdIgl2. Most EdIgl1 and EdIgl2 proteins were co-localized on the surface and in the cytoplasm of trophozoites, based on confocal microscopy. However, a different localization of EdIgl1 and EdIgl2 in intracellular vacuoles and a different level of phenotypic expression of the two Igls were also observed. These results demonstrate that Igls are important proteins even in non-pathogenic amoeba and that Igl1 and Igl2 may possess different functions.

An Entamoeba sp. strain, P19-061405, was isolated from a rhesus monkey in Nepal and characterized genetically. The strain was initially identified as Entamoeba histolytica using PCR amplification of peroxiredoxin genes. However, sequence analysis of the 18S rRNA gene showed a 0.8% difference when compared to the reference E. histolytica HM-1:IMSS human strain. Differences were also observed in the 5.8S rRNA gene and the internal transcribed spacer (ITS) regions 1 and 2, and analysis of the serine-rich protein gene from the monkey strain showed unique codon usages compared to E. histolytica isolated from humans. The amino acid sequences of two hexokinases and two glucose phosphate isomerases also differed from those of E. histolytica. Isoenzyme analyses of these enzymes in the monkey strain showed different electrophoretic mobility patterns compared with E. histolytica isolates. Analysis of peroxiredoxin genes indicated the presence of at least seven different types of protein, none of which were identical to proteins in E. histolytica. When the trophozoites from the monkey strain were inoculated into the livers of hamsters, formation of amebic abscesses was observed 7 days after the injection. These results demonstrate that the strain is genetically different from E. histolytica and is virulent. Revival of the name Entamoeba nuttalli is proposed for the organism.

The intercalation behavior of cationic porphyrin derivatives within the interlayer spaces of nano-layered clay minerals has been investigated. The porphyrins were successfully intercalated by the newly adopted method of repeated freeze-thaw cycles. The absorption spectra of the porphyrins were compared in the solution phase, adsorbed onto the exfoliated clay nano-sheets, intercalated within the interlayer spaces of clay sheets dispersed in water and intercalated in dry films. Substantial red shifts of the λmax values in the absorption spectra of the porphyrins were observed on the exfoliated clay sheets, and further red shifts were induced within the interlayer space. The dry films of the intercalated samples exhibited the largest red shifts. X-ray diffraction studies revealed that the clearance space between the layers in these intercalated hybrid compounds is only large enough for the porphyrins to be rigidly packed parallel to the clay layer. For the exfoliated clay nano-sheets, theoretical calculations were carried out on the correlation between the dihedral angle of the meso-substituted pyridiniumyl plane vs. the porphyrin ring and the λmax of the porphyrin Soret band. An extrapolation of the experimental λmax value to the correlation curve, afforded the dihedral angle to be 61.6°. The microscopic structure of the adsorbed state of the cationic porphyrins on the exfoliated clay nano-sheets was, thus, proposed to involve an orientation parallel to the clay surface, with a distance of 0.15 nm from the surface, which implies the expulsion of the solvent water molecules.

Complexes formed with synthetic clay minerals and dicationic and tetracationic porphyrins were prepared. The Soret bands of the multicationic porphyrins shifted to longer wavelength upon the format...

Unique species of macaques are distributed across Sulawesi Island, Indonesia, and the details of Entamoeba infections in these macaques are unknown. A total of 77 stool samples from Celebes crested macaques (Macaca nigra) and 14 stool samples from pigs were collected in Tangkoko Nature Reserve, North Sulawesi, and the prevalence of Entamoeba infection was examined by PCR. Entamoeba polecki was detected in 97% of the macaques and all of the pigs, but no other Entamoeba species were found. The nucleotide sequence of the 18S rRNA gene in E. polecki from M. nigra was unique and showed highest similarity with E. polecki subtype (ST) 4. This is the first case of identification of E. polecki ST4 from wild nonhuman primates. The sequence of the 18S rRNA gene in E. polecki from pigs was also unique and showed highest similarity with E. polecki ST1. These results suggest that the diversity of the 18S rRNA gene in E. polecki is associated with differences in host species and geographic localization, and that there has been no transmission of E. polecki between macaques and pigs in the study area.

We have proposed the revival of the name Entamoeba nuttalli for a virulent ameba strain, P19-061405, from a rhesus macaque and located it phylogenetically between E. histolytica and E. dispar. As E. nuttalli was originally described for an ameba found in a toque macaque in Sri Lanka, the prevalence and characteristics of Entamoeba species in wild toque macaques were examined. PCR analysis of 227 stool samples from six locations showed positive rates for E. nuttalli, E. dispar, and E. histolytica of 18.5%, 0.4%, and 0%, respectively. Fifteen E. nuttalli strains were cultured successfully from five locations. The 18S ribosomal RNA gene showed only three nucleotide differences in comparison with P19-061405 strain. In isoenzyme analysis, the pattern of hexokinase in Sri Lankan strains was different from that of P19-061405 strains and the difference was confirmed by analysis of the genes. Hepatic inoculation of one of the Sri Lankan E. nuttalli strains in hamsters resulted in amebic abscess formation and body weight loss. These results demonstrate that E. nuttalli is prevalent in wild toque macaques and that several characteristics of the strains are unique. We conclude that use of the name E. nuttalli is appropriate for the new Entamoeba species found in nonhuman primates.

Cryptosporidiosis has been reported to be associated with HIV/acquired immune deficiency syndrome, which greatly reduces the quality of life and shortens the life expectancy of HIV-infected patients. In order to properly treat the infected patients, accurate and automatic diagnostic tools need to be developed. In this study, a novel microfluidic immunochip system was presented for the surveillance and the rapid detection of Cryptosporidium infection in 190 HIV-infected patients from Guangxi, China, using the P23 antigen of Cryptosporidium. The procedure of detection can be completed within 10 min with 2 μl sample consumption. The system also was evaluated using the standard ELISA method. Among 190 HIV-infected individuals, the rate of P23 positivity was 13.7%. Seropositivity in HIV-infected individuals was higher in female patients. The seropositivity to P23 was higher in HIV-infected individuals with high viral load, although the difference was statistically insignificant. Significantly higher Cryptosporidium seropositivity was observed in HIV-infected individuals with a CD4(+) T-cell count of200 cells/μl than in those with ≥200 cells/μl. Our results also demonstrate that a lower CD4(+) T-cell count may reflect an increased accumulated risk for cryptosporidiosis. The detection system was further validated using the standard ELISA method and good correlation between the two methods was found (r = 0.80). Under the same sensitivity, this new microfluidic chip device had a specificity of 98.2%. This developed system may provide a powerful platform for the fast screening of Cryptospordium infection in HIV-infected patients.

A unique guest-guest ion exchange method was developed for preparing a thin film of a nano-layered K(4)Nb(6)O(17).3H(2)O that possesses both (1) optical transparency and (2) ion-exchangeability under ambient conditions without calcination at high temperature. An optically transparent Ru(bpy)(3)(2+)-K(4)Nb(6)O(17) hybrid thin film, a photoresponsive electrode, was successfully prepared by the guest-guest exchange method by use of the intercalation compound MV(2+)-K(4)Nb(6)O(17) as a precursor. The optically transparent Ru(bpy)(3)(2+)-K(4)Nb(6)O(17) hybrid thin films have been characterized by X-ray diffraction, SEM, AFM, IR, and UV spectroscopies, as well as elemental analysis. The electrochemical behavior of the ITO/Ru(bpy)(3)(2+)-K(4)Nb(6)O(17) hybrid thin film electrode was studied; it also exhibits swift photoresponse in the visible region.

Novel inorganic/organic hybrids formed from cationic porphyrins and synthetic clay were investigated. Di-, tri- and tetra-cationic charged porphyrins with different distances between adjacent cationic sites within the molecules were incorporated into the hybrids with synthetic clay. When tetra-cationic porphyrins were used, in which distances between adjacent cation sites were 1.05 and 1.31 nm, the porphyrin molecules adsorb on the clay surfaces with high density, neutralizing all of the negative charges of the clay surface, i.e. 100% adsorption vs. the cation exchange capacity (CEC), without aggregation, even though it is generally difficult to control or suppress the aggregation of organic molecules on inorganic surfaces. The mean center-to-center distance between porphyrin molecules in the hybrids was estimated to be 2.4 nm. When Δ l (the difference between the inter-cationic charge distance in the porphyrin molecule and the inter-anionic charge distance on the clay surface) was larger than ca. 0.2 nm, the porphyrin molecules adsorbed in amounts smaller than 100% vs. CEC. In the cases of di- and tri-cationic charged porphyrins, similar adsorption behavior was observed. The formation of these unique hybrids was rationalized by a size-matching of distances between the charged sites in the porphyrin molecule and on the clay surface (‘size-matching rule’).

We have recently identified a 150-kDa surface antigen of Entamoeba histolytica as an intermediate subunit (Igl) of galactose- and N -acetyl -d- galactosamine-inhibitable lectin, which is a cysteine-rich protein consisting of 1,101 amino acids (aa) and containing multiple CXXC motifs in amino acid sequences. In the present study, full-length Igl except for the signal sequences (aa 14 to 1088) and three fragments of Igl—the N-terminal part (aa 14 to 382), the middle part (aa 294 to 753), and the C-terminal part (aa 603 to 1088)—were prepared in Escherichia coli , and the reactivity of these recombinant proteins with sera from patients with amebiasis was examined by means of enzyme-linked immunosorbent assay (ELISA). Sera from 57 symptomatic patients with amebic liver abscess or amebic colitis, sera from 15 asymptomatic cyst passers, sera from 40 individuals with other protozoan infections, and sera from 50 healthy controls were used. The sensitivity and specificity of the recombinant full-length Igl in the ELISA were 90 and 94%, respectively. When three fragments were used as antigens in the ELISA, the sensitivities were 56% in the N terminus, 92% in the middle part, and 97% in the C terminus. The specificities of the three antigens were 96% in the N terminus and 99% in both the middle and C-terminal fragments. These results demonstrate that Igl is well recognized in not only symptomatic but also asymptomatic patients with E. histolytica infection and that the carboxyl terminus of Igl is an especially useful antigen for the serodiagnosis of amebiasis.

A combinatorial human immunoglobulin gene library was constructed from peripheral lymphocytes of an asymptomaticEntamoeba histolyticacyst passer and screened for the production of Fab antibody to the parasite. One of the Fab clones, CP33, recognized the 260-kDa galactose- andN-acetyl-d-galactosamine (Gal/GalNAc)-specific lectin ofE. histolytica. By shuffling the heavy and light chains of CP33 with the heavy and light chains of two libraries derived from the cyst passer and a liver abscess patient, 18 additional clones were obtained. Sequence analysis of the heavy-chain genes, including CP33-H, revealed that all the nearest V-segment germ lines belonged to the VH3 family (VH3-21, VH3-30, VH3-48, and VH3-53), but the levels of homology were only 85 to 95%. The closest D-segment germ line was D2-2 or D6-6, and for the J-segment the closest germ line was JH4b or JH6b. On the other hand, all the light-chain genes, including CP33-L, belonged to the Vκ1 family, in which the closest Vκ germ line gene was 02/012 or L5, with the Jκ1, Jκ2, Jκ4, or Jκ5 segment. CP33 and three other Fabs obtained by light-chain shuffling were purified and analyzed further. All of these Fabs recognized the cysteine-rich domain of the 170-kDa heavy subunit of the Gal/GalNAc lectin. Preincubation ofE. histolyticatrophozoites with these Fabs significantly inhibited amebic adherence to Chinese hamster ovary cells and also inhibited erythrophagocytosis. The ability of the neutralizing antibodies to block erythrophagocytosis for the first time implicates the lectin in phagocytosis and VH3 antibodies in defense against parasitic infections. These results demonstrate the utility of a combinatorial human immunoglobulin gene library for identifying and characterizing neutralizing antibodies from humans with amebiasis.

Intercalation of seven types of cationic surfactants, including polyfluorinated surfactants, into a cation-exchangeable clay mineral, saponite, was investigated. All of the surfactants were found to intercalate in amounts exceeding the cation exchange capacity (CEC). This tendency was more evident in the cases of the polyfluorinated surfactants, ((((perfluoroalkanoyl)amino)ethyl)hexadecyl)dimethylammonium bromide (CnF-S, where n indicates the number of carbons in the perfluoroalkyl group, F denotes a fluorinated surfactant, and S denotes a surfactant having a single, long alkyl chain); C3F-S exhibited intercalation up to 4.4 times CEC as a saturated adsorption limit. The saponite interlayer distance increased upon surfactant intercalation and reached a constant value at intercalation levels exceeding CEC. The occupied areas for each surfactant molecule decreased in the order C1F-S > C2F-S > C3F-S among the polyfluorinated surfactants. All of the experimental results, including the saturated intercalation ...

An experimental study has been performed on the ignition process of liquid fuel particles to obtain the fundamental data of spray combustion. The particles consisted of the center particle and ligands arranged in a geometric configuration. The number of ligands was defined as coordination number. The particles were set in a combustion chamber and the combustion chamber was filled with a propane-air mixture at atmospheric pressure. The propagating laminar flame was formed with the hot wire ignition and the particles were ignited by the flame. The results showed that in case of coordination number 3, the dimensionless ignition delay of the center particle decreased with a decrease in the dimensionless particle distance, and that in case of coordination number 4 and 6, the dimensionless ignition delay of the center particle had a minimum. The minimum was the smallest in case of coordination number 6. The smaller particle diameter had the larger dimensionless particle distance that showed the minimun dimensionless ignition delay.

We developed an immunochromatographic assay kit that uses fluorescent silica nanoparticles bound to anti- Acanthamoeba antibodies (fluorescent immunochromatographic assay [FICGA]) and evaluated its efficacy for the detection of Acanthamoeba and diagnosis of Acanthamoeba keratitis (AK). The sensitivity of the FICGA kit was evaluated using samples of Acanthamoeba trophozoites and cysts diluted to various concentrations. A conventional immunochromatographic assay kit with latex labels (LICGA) was also evaluated to determine its sensitivity in detecting Acanthamoeba trophozoites. To check for cross-reactivity, the FICGA was performed by using samples of other common causative pathogens of infectious keratitis, such as Pseudomonas aeruginosa , Staphylococcus aureus , Staphylococcus epidermidis , and Candida albicans . Corneal scrapings from patients with suspected AK were tested with the FICGA kit to detect the presence of Acanthamoeba , and the results were compared with those of real-time PCR. The FICGA kit detected organisms at concentrations as low as 5 trophozoites or 40 cysts per sample. There were no cross-reactivities with other pathogens. The FICGA was approximately 20 times more sensitive than the LICGA for the detection of Acanthamoeba trophozoites. The FICGA kit yielded positive results for all 10 patients, which corresponded well with the real-time PCR results. The FICGA kit demonstrated high sensitivity for the detection of Acanthamoeba and may be useful for the diagnosis of AK.

Acanthamoeba sp. parasites are the causative agents of Acanthamoeba keratitis, fatal granulomatous amoebic encephalitis, and cutaneous infections. However, there are currently no effective drugs for these organisms. Here, we evaluated the activity of the antimalarial agent artemether against Acanthamoeba castellanii trophozoites and identified potential targets of this agent through a proteomic approach. Artemether exhibited in vitro amoebicidal activity in a time- and dose-dependent manner and induced ultrastructural modification and cell apoptosis. The iTRAQ quantitative proteomic analysis identified 707 proteins that were differentially expressed after artemether treatment. We focused on phosphoglycerate dehydrogenase and phosphoserine aminotransferase in the serine biosynthesis pathway because of their importance to the growth and proliferation of protozoan and cancer cells. The expression of these proteins in Acanthamoeba was validated using quantitative real-time PCR and Western blotting after artemether treatment. The changes in the expression levels of phosphoserine aminotransferase were consistent with those of phosphoglycerate dehydrogenase. Therefore, the downregulation of phosphoserine aminotransferase may be due to the downregulation of phosphoglycerate dehydrogenase. Furthermore, exogenous serine might antagonize the activity of artemether against Acanthamoeba trophozoites. These results indicate that the serine biosynthesis pathway is important to amoeba survival and that targeting these enzymes would improve the treatment of Acanthamoeba infections. Artemether may be used as a phosphoglycerate dehydrogenase inhibitor to control or block Acanthamoeba infections.

A retrospective cohort study was conducted in 55 symptomatic patients with amebic colitis that visited at St. Luke's International Hospital and Mie University Hospital from 1994 through 2013. To diagnose amebic colitis, 40 patients underwent total colonoscopy within 1 week after hospital visiting and before receiving any treatment. The percentage of characteristic endoscopic findings of amebic colitis including discrete ulcers or erosions with white or yellow exudates were 0% in terminal ileum, 93% in cecum, 28% in ascending, 25% in transverse, 15% in descending, 20% in sigmoid colon and 45% in rectum. The rectal lesions in 55% of patients with amebic colitis were nonspecific. The trophozoite identification rate by direct smear of intestinal tract washings performed during colonoscopy was 88%. The protozoan identification rate was 70% in biopsy specimens taken from the periphery of the characteristic discrete ulcers. Total colonoscopy should be considered for the diagnosis of amebic colitis.

Today, amebiasis is commonly found in nearly all tropical and subtropical countries. Improved diagnostic tests are beginning to overturn some of the commonly held beliefs about amebiasis. Amebiasis outbreaks in developed nations have resulted from contamination of water supplies with sewage secondary to inadequate water maintenance and treatment. Four glycolytic isoenzymes (glucosephosphate isomerase, phosphoglucomutase, hexokinase, and malic enzyme) exhibit different migration patterns on gel electrophoresis. The cytologic diagnosis of amebiasis is neither specific nor sensitive. Entamoeba histolytica and Entamoeba dispar are identical in appearance. Since in most areas E. dispar is more common than E. histolytica, this makes cytologic diagnosis extremely nonspecific. Detection of the Gal-GalNAc lectin antigen in serum can be used to identify E. histolytica infection. The application of riboprinting techniques may therefore provide a better understanding of the epidemiology of amebiasis, including different modes of transmission. One study investigated 105 cases of travelers returning from regions of endemicity and residents of those regions, with infection with E. histolytica and/or E. dispar identified by microscopic detection. Twenty-one of 60 isolates were identified as E. histolytica by PCR and hexokinase isoenzyme, including isolates from 13 of 26 infected patients with amebiasis-like symptoms. The other important group to affect the epidemiology of amebiasis in the United States is the immigrant population, especially Hispanic and Pacific Islander immigrants and Indochinese refugees.

A 35-year-old Japanese man had an intermittent fever and mild headache for eight weeks after he returned to Japan from working in Mozambique. He had taken antimalarial prophylaxis (doxycycline) for 25 weeks, and stopped taking this drug two weeks after his return. Microscopic examination of a peripheral blood smear showed a mixed infection with Plasmodium vivax, P. falciparum, and P. ovale. In addition, a nested polymerase chain reaction and subsequent sequencing detected specific DNA sequences of four species of Plasmodium, including P. malariae. The patient was successfully treated with artemether-lumefantrine and primaquine phosphate. The present case is a rare instance of a mixed infection with four species of Plasmodium. Nonimmune persons in malaria-endemic areas may have a risk of mixed infection. All four species must be identified by using sensitive and specific tests, such as a nested polymerase chain reaction, in addition to conventional morphologic identification.

Cryptosporidium oocysts, morphologically identified as Cryptosporidium parvum, were isolated from 22 human and 14 bovine cases in Japan, and were genotyped by means of a PCR/RFLP analysis of the polythreonine gene. DNA profiles of human isolates gave three distinct genotypes, namely an anthroponotic genotype 1, zoonotic genotype 2 and a new genotype. Isolates from bovine samples gave zoonotic genotype 2. The unusual genotype of Cryptosporidium was isolated from the feces of three immunologically healthy adults, and was further characterized by the sequence analysis of the 18S rRNA gene. The third genotype was identified as Crypto sporidium meleagridis, demonstrating that C. meleagridis, which occurs worldwide, has the potential to infect humans regardless of their immunological condition.

Killing by Entamoeba histolytica requires parasite adherence to host galactose- and N -acetyl- d -galactosamine (Gal/GalNAc)-containing cell surface receptors. A 260-kDa heterodimeric E. histolytica Gal/GalNAc lectin composed of heavy (Hgl) and light (Lgl) subunits has been previously described. Here we present the cloning and characterization of Igl, a 150-kDa intermediate subunit of the Gal/GalNAc lectin. Igl, Hgl, and Lgl colocalized on the surface membrane of trophozoites. Two unlinked copies of genes encoding Igl shared 81% amino acid sequence identity (GenBank accession no. AF337950 and AF337951 ). They encoded cysteine-rich proteins with amino- and carboxy-terminal hydrophobic signal sequences characteristic of glycosylphosphatidylinositol (GPI)-anchored membrane proteins. The igl genes lacked carbohydrate recognition domains but were members of a large family of amebic genes containing CXXC and CXC motifs. These data indicate that Igl is part of the parasite's multimolecular Gal/GalNAc adhesin required for host interaction.

A monoclonal antibody, EH3015, prevents in vitro adherence of Entamoeba histolytica trophozoites to mammalian cells and inhibits amebic liver abscess formation in hamsters. By immunoaffinity chromatography with the monoclonal antibody, purified E. histolytica antigens with molecular masses of 150 and 170 kDa under non-reduced conditions were obtained. Hamsters were immunized with these antigens (group I) or with fractions further purified by polyacrylamide gel electrophoresis (group II). Pooled immune sera from the two groups inhibited in vitro amebic adherence to Chinese hamster ovary cells by 98% at 1:10 dilutions. The immunized hamsters were challenged by the intrahepatic injection of E. histolytica trophozoites. Complete protection from abscess formation was observed in 38% of hamsters in group I and 67% in group II, whereas all control hamsters inoculated only with adjuvant developed amebic liver abscesses. In the immunized hamsters, the abscesses in the two groups were significantly smaller than in the controls. These results demonstrate that the E. histolytica antigens are possible vaccine candidates for amebiasis.

A total of 268 nonhuman primates (20 species) kept in the Primate Research Institute, Kyoto University, Japan, were surveyed for intestinal amebas. Total positive rates as based on the presence of cysts in the stool following formalin-ether sedimentation were as follows: Entamoeba histolytica/E. dispar, 53%; E. coli, 34%; E. hartmanni, 34%; Iodamoeba buetschlii, 25%; Endolimax nana, 8%; and E. chattoni, 3%. Positive rates were higher in Old World monkeys and lower in New World monkeys. All the 141 E. histolytica/E. dispar-positive animals were Macaca monkeys. The E. histolytica/E. dispar-positive samples were analyzed by polymerase chain reaction (PCR) for identification of E. histolytica and E. dispar. E. dispar DNA was detected in 137 samples, whereas no E. histolytica DNA was seen. Zymodeme analysis and reactivity to monoclonal antibodies of cultured trophozoites also supported the presence of E. dispar and the absence of E. histolytica. When the sera of 93 macaques were examined by an indirect fluorescent antibody test, only 3 animals proved to be positive for E. histolytica, showing the lowest titer. These results demonstrate that infection with E. dispar, but not E. histolytica, is predominant in macaques.

Solvent and substituent effects on the competition between internal conversion and triplet formation were studied systematically for aminofluorenones and their N-methylated derivatives. Intersystem crossing (ISC) was found to be the dominant process for the singlet excited 1-amino- and 1-methylaminofluorenone in all solvents. The short fluorescence decay time of these compounds does not originate from intramolecular hydrogen bonding induced internal conversion but it is due to the fast triplet formation. Rather slow (kISC⩽4.8 × 107 s−1) and solvent insensitive intersystem crossing characterizes the photophysical behavior of 2-, 3- and 4-aminofluorenones but their internal conversion rate strongly increases with solvent polarity. The change of the internal conversion rate constants with molecular structure and solvent can be rationalized in terms of the energy gap law.

We report here an efficient route to the synthesis of highly fluorescent hexaaryl[3]radialenes using the oligomerization of ate-type copper carbenoids, followed by cyclization with hexamethylditin and Pd(PPh3)4; the structures of the [3]dendralene and hexaaryl[3]radialenes were determined by X-ray crystallographic analysis.

Tachibana, H., and Cheng, X.-J. 2000. Entamoeba dispar: Cloning and Characterization of Peroxiredoxin Genes. Experimental Parasitology94, 51–55.

Genes coding for human antibody Fab fragments specific for Entamoeba histolytica were cloned and expressed in Escherichia coli . Lymphocytes were separated from the peripheral blood of a patient with an amebic liver abscess. Poly(A) + RNA was isolated from the lymphocytes, and then genes coding for the light chain and Fd region of the heavy chain were amplified by a reverse transcriptase PCR. The amplified DNA fragments were ligated with a plasmid vector and were introduced into Escherichia coli . Three thousand colonies were screened for the production of antibodies to E. histolytica HM-1:IMSS by an indirect fluorescence-antibody (IFA) test. Lysates from five Escherichia coli clones were positive. Analysis of the DNA sequences of the five clones showed that three of the five heavy-chain sequences and four of the five light-chain sequences differed from each other. When the reactivities of the Escherichia coli lysates to nine reference strains of E. histolytica were examined by the IFA test, three Fab fragments with different DNA sequences were found to react with all nine strains and another Fab fragment was found to react with seven strains. None of the four human monoclonal antibody Fab fragments reacted with Entamoeba dispar reference strains or with other enteric protozoan parasites. These results indicate that the bacterial expression system reported here is effective for the production of human monoclonal antibodies specific for E. histolytica . The recombinant human monoclonal antibody Fab fragments may be applicable for distinguishing E. histolytica from E. dispar and for use in the serodiagnosis of amebiasis.

We used the polymerase chain reaction (PCR) to study the distribution of Entamoeba histolytica and E. dispar in 1,872 individuals in 14 communities in the northern Philippines. Here we report a field study using a DNA extraction protocol from formalin-fixed stool specimens as previously reported. This assay detected 137 stools (7.318%) containing E. dispar and 18 stools (0.961%) containing E. histolytica. The most affected age group for E. histolytica/E. dispar infections were those 5-14 years of age. There was no significant difference in the sex distribution of E. histolytica, while in the case of E. dispar, a higher prevalence was observed in females (9.186%) than in males (5.731%) (P < 0.01). An apparent clustering of stool-positive cases of E. histolytica and E. dispar was also observed in the northern part of the study area. The results of this survey demonstrate that E. dispar is highly prevalent in the communities studied. Moreover, it offers promise for the PCR using DNA extracted from formalin-fixed stools as a sensitive epidemiologic tool for detecting E. histolytica and E. dispar infections., American Journal of Tropical Medicine and Hygiene, 59(6), pp.916-921; 1998

A monoclonal antibody that reacts with a 150-kDa protein of Entamoeba histolytica on Western immunoblotting under nonreducing conditions inhibits the adherence and cytotoxicity of the ameba to mammalian cells in vitro. Affinity purification of solubilized trophozoites using the monoclonal antibody and electrophoresis yielded three glycoproteins with molecular masses of 150, 170, and 260 kDa, suggesting the existence of either a common epitope or the close association of these proteins. The 260-kDa fraction was identified as the well-known galactose (Gal)- and N-acetyl-D-galactosamine (GalNAc)-inhibitable lectin. The 150- and 170-kDa fractions seemed to exist as part of a 380-kDa native protein with an isoelectric point of pH 6.9. The N-terminal amino acid sequence of the 150-kDa protein was unique, indicating that the protein was not a degraded product of the 260-kDa lectin. By gel filtration, the 260-kDa lectin and the 150/170-kDa protein could be separated. When Chinese hamster ovary cells were pretreated with the fraction consisting of the 150/170-kDa protein the adherence of trophozoites to Chinese hamster ovary cells was competitively inhibited to a level equivalent to that observed for the 260-kDa lectin. The inhibitory effect was lost in the presence of Gal and GalNAc but was not influenced by the presence of glucose. These results demonstrate that the 150/170-kDa protein is a Gal/GalNAc-inhibitable lectin. The existence of a sugar-binding domain in the protein was confirmed by Gal-affinity chromatography.

Four isolates of Entamoeba dispar identified by their hexokinase and phosphoglucomutase isoenzyme profile and by their failure to react with Entamoeba histolytica-specific monoclonal antibody (4G6) could be grown in either Diamond's BI-S-33 medium, newly developed BCSI-S (Biosate cysteine starch iron-serum) medium, or casein-free YI-S medium in the presence of Crithidia fasciculata (ReF-1:PRR) sterilized by heating 56 degrees C for 30 min and subsequent incubation with 1% hydrogen peroxide for 24 hours at 4 degrees C. After the cultures were maintained for over 50 passages, the amebae were identified as E. dispar by isoenzyme analysis, polymerase chain reaction with E. histolytica- and E. dispar-specific primers, i.e. p11 plus p12 and p13 plus p14, respectively, and by negative reactivity with monoclonal antibody 4G6. The flagellates added to the culture were judged to be metabolically inactive based on the results of nuclear magnetic resonance spectroscopy, electron microscopy, and polarographic analysis. All of these findings suggest that E. dispar can grow in vitro with metabolically inactive C. fasciculata as a culture associate.

Fourteen monoclonal antibodies (mAbs) were produced against a strain of Acanthamoeba castellanii isolated from a human cornea. The reactivity of the mAbs to reference strains of Acanthamoeba was examined by an indirect fluorescence antibody test (IFA) and Western immunoblot analysis. Nine mAbs reacted specifically with a known pathogenic reference strain of A. castellanii, but not with a non-pathogenic strain or other Acanthamoeba spp. The antigen recognized by these mAbs had a molecular mass of 17 kDa. The remaining five mAbs reacted with A. castellanii and A. polyphaga, members of group II (Pussard and Pons) but not with A. astronyxis (group I) or A. culbertsoni (group III). Western immunoblot analysis revealed that the latter mAbs stained many protein bands ranging from 30 to 150 kDa. None of the 14 mAbs reacted with Naegleria gruberi, N. fowleri, or Entamoeba histolytica. These observations suggest that an antigen common in group II as well as a pathogenic A. castellanii-specific antigen are present. Slot blot reactivity was comparable to the IFA. Under certain circumstances, therefore, slot blot analysis with a panel of mAbs should be helpful in the detection of keratitis-producing strains of Acanthamoeba.