Exploring the size of reference population for expected accuracy of genomic prediction using simulated and real data in Japanese Black cattle.

BACKGROUND: Size of reference population is a crucial factor affecting the accuracy of prediction of the genomic estimated breeding value (GEBV). There are few studies in beef cattle that have compared accuracies achieved using real data to that achieved with simulated data and deterministic predictions. Thus, extent to which traits of interest affect accuracy of genomic prediction in Japanese Black cattle remains obscure. This study aimed to explore the size of reference population for expected accuracy of genomic prediction for simulated and carcass traits in Japanese Black cattle using a large amount of samples. RESULTS: A simulation analysis showed that heritability and size of reference population substantially impacted the accuracy of GEBV, whereas the number of quantitative trait loci did not. The estimated numbers of independent chromosome segments (Me) and the related weighting factor (w) derived from simulation results and a maximum likelihood (ML) approach were 1900-3900 and 1, respectively. The expected accuracy for trait with heritability of 0.1-0.5 fitted well with empirical values when the reference population comprised > 5000 animals. The heritability for carcass traits was estimated to be 0.29-0.41 and the accuracy of GEBVs was relatively consistent with simulation results. When the reference population comprised 7000-11,000 animals, the accuracy of GEBV for carcass traits can range 0.73-0.79, which is comparable to estimated breeding value obtained in the progeny test. CONCLUSION: Our simulation analysis demonstrated that the expected accuracy of GEBV for a polygenic trait with low-to-moderate heritability could be practical in Japanese Black cattle population. For carcass traits, a total of 7000-11,000 animals can be a sufficient size of reference population for genomic prediction.

Mandible fractures and dental injuries related to road traffic accidents over a 12-year period-Retrospective multicentre study.

BACKGROUND/AIM: Road traffic accidents (RTAs) are a common cause of maxillofacial injuries. The aim of this retrospective multicentre study was to investigate the characteristics of maxillofacial fractures and dental injuries that occurred in RTAs in Miyagi, Japan. MATERIALS AND METHODS: The records of 404 patients with maxillofacial injuries treated at the Oral and Maxillofacial Surgery Departments of four different institutions over a period of 12 years were analysed. Ninety-nine of these patients had suffered these injuries in an RTA. RTA-related cases were divided according to age, gender, presentation month, presentation day of the week, transportation mode, time of accident, fracture sites and fracture mechanism. RESULTS: There were 72 males and 27 females who suffered injuries as the result of an RTA, for a male-to-female ratio of 2.7:1.0, with a mean age of 35.3 years (range, 1-86 years old). Most of the accidents occurred in June and on a Wednesday, and most of the affected patients were riding a bicycle at the time. The number of patients with maxillofacial injuries related to bicycle riding showed an increasing trend in recent years. Mandible fractures were the most prevalent, followed by dental injuries and maxilla fractures. In cases with a single fracture of the mandible, the symphysis was the most frequent site, while in those with multiple fractures, the association of symphysis and bi-lateral condyle fractures was the greatest. For bicycle-related accidents, a single fracture in the mandible occurred more often than multiple fractures. CONCLUSIONS: The number of RTA-related injuries while bicycle riding showed an increasing trend with mandible fractures commonly seen in those cases. Efforts to reduce maxillofacial injuries related to bicycle accidents are needed.

Mandibular fractures and dental injuries sustained during baseball and softball over 14 years in a Japanese population: A retrospective multicentre study.

BACKGROUND/AIM: Active participation in sports is a risk factor for maxillofacial fractures. The aim of this retrospective multicentre study was to survey and evaluate the characteristics of mandibular fractures, and dental injuries that occurred during the practice of baseball and softball in Sendai, Japan. MATERIAL AND METHODS: The records of 454 patients with maxillofacial fractures from three departments of Oral and Maxillofacial Surgery across a period 14 years were analysed. Fifty-one patients with 56 mandible fractures and dental injuries that occurred playing baseball or softball were included in this multicenter retrospective study. Patients were divided according to age, gender, sites of fractures, mechanism of fractures and treatment methods. RESULTS: There were 42 males and nine females, with a male-to-female ratio of 4.7:1.0. The mean age was 19.9 years old (range: 13-47 years old). As for the site, body of the mandible fractures prevailed, followed by the condyle, symphysis and angle. Fractures were mostly caused by the impact of a ball (42; 82.4%), followed by collisions with another player (5; 9.8%) and direct strike of a bat (4; 7.8%). All patients with mandibular fractures were treated with open reduction and internal fixation, except for six patients with condylar head fractures who were managed conservatively. CONCLUSIONS: The impact of a thrown ball against the batter's mandible can cause a condylar fracture when playing baseball and softball.

Phylogeography of Endangered Bitterling Acheilognathus melanogaster Endemic to Eastern Japan.

The bitterling Acheilognathus melanogaster is a critically endangered primary freshwater fish endemic to the Pacific side of eastern Japan. To elucidate A. melanogaster genetic structure, we investigated phylogeography in nine populations, using gene sequences of mitochondrial Cytochrome b (Cytb), as well as nuclear Rhodopsin (Rho) and glycosyltransferase (Glyt). We found four Cytb-based geographical clusters unevenly divided between the northern and southern regions, with smaller groups in the south. Of the nuclear genes, Glyt did not show geographical differentiation, whereas Rho formed two clusters: one widely occurring and another restricted to central regions. Genetic diversity was generally higher in southern than in northern populations. Our results suggest that conservation of southern local populations is particularly important in maintaining the genetic diversity of this endangered fish.

[Two cases of Paragonimiasis westermani in a Chinese family diagnosed with the Ouchterlony double diffusion method].

We report two cases of Paragonimus westermani infection in a Chinese family in Japan. A 41-year-old husband and his 40-year-old wife were infected with P. westermani after consuming a homemade Chinese traditional "Drunken Crab." They were a family with two children who had lived in Japan for 19 years. The crabs were Eriocheir japonica sent from the Kyusyu area that they had pickled at home with soy sauce and Chinese liquor for 5 days. Their children did not eat any of the crabs. One month after consuming the crabs, the husband came to our outpatient clinic with fever and chest pain and his wife also presented with a persistent cough. Both patients had a high peripheral blood eosinophil count (husband:18,900/µL, wife:10,600/µL) with pulmonary effusion, nodular shadow, and pneumothorax in chest X-ray findings. Paragonimiasis was suspected from the episode of consuming the crabs. No parasite eggs were seen in their sputum and stool samples. A multiple-dot ELISA was performed with the sera to screen for parasitic infections, but the result was only weakly positive for P. westermani antigen in the husband and a slightly positive reaction in the wife. The diagnosis of P. westermani was achieved with the double diffusion Ouchterlony method using P. westermani antigen and P. miyazakii antigen. Praziquantel administration for three days improved the symptoms in both patients. The Ouchterlony method proved useful in diagnosing paragonimiasis in these cases.

Expression analysis of Entamoeba invadens profilins in encystation and excystation.

Cell motility by actin cytoskeleton is essential for differentiation processes of excystation and encystation of Entamoeba. We recently studied an actin depolymerizing factor (ADF)/cofilin (Cfl) of Entamoeba invadens (Ei), and demonstrated its contribution to the encystation and excystation of E. invadens through actin cytoskeletal reorganization. Profilin is also an actin-binding protein but its function is different from that of Cfl in actin assembly. This study investigated E. invadens profilins in relation to encystation and excystation which were induced in axenic culture systems. A homology search of the E. invadens genome database and molecular cloning identified four profilins of the parasite named EiPFN1, EiPFN2, EiPFN3, and EiPFN4. There were also multiple genes of profilin in Entamoeba histolytica (Eh) and Entamoeba dispar (Ed), each of which had three profilins. A search for conserved domains revealed that these profilins of Entamoeba had actin, phosphoinositide, and poly-proline binding sites. Phylogenetic analysis revealed that EiPFN3 and EiPFN4 formed the same clades including EhPFN3 and EdPFN3, and EhPFN2 and EdPFN2, respectively, while EiPFN1 and EiPFN2 were separated from EhPFN1 and EdPFN1. Rabbit anti-EiPFN1 serum reacted with recombinant EiPFN3 and EiPFN4 but not EiPFN2, and also reacted with EiPFN in lysates of cysts and trophozoites. Immunofluorescence staining with this antiserum showed co-localization of EiPFN with actin beneath the cell membrane through the life stages and also showed cytoplasmic localization. Both proteins proved to be rich in pseudopodia of trophozoites. Real-time RT-PCR showed that the mRNA level of EiPFN1 and EiPFN4 in trophozoites was comparable but that of EiPFN2 and EiPFN3 was very low. During encystation, the mRNA expression of EiPFN1 and EiPFN4 increased remarkably in the early phase much higher than that of EiPFN2 and EiPFN3. Then, the expression of all four PFNs sharply decreased in the later phase. This was in contrast to the sharp decrease in the mRNA level of EiCfl-2 during encystation in our previous study. In cysts, EiPFN1 was most abundantly expressed and EiPFN4 was at a lower level, while the expressions of EiPFN2 and EiPFN3 were virtually absent. Following the induction of excystation, mRNA levels of EiPFN1, EiPFN2, and EiPFN4 in cysts 5 h after induction were significantly higher than those in cysts before induction, while that of EiPFN3 was slightly higher than before induction. The mRNAs of EiPFN1 increased most extensively when the excystation was induced in the presence of cytochalasin D. Small interfering RNA (siRNA) to EiPFN1 inhibited both encystation and excystation but not growth. These findings demonstrate different expression of EiPFNs and the contribution of EiPFN to the encystation and excystation.

Entamoeba invadens: identification of ADF/cofilin and their expression analysis in relation to encystation and excystation.

The differentiation processes of excystation and encystation of Entamoeba are essential for infection and completion of their life-cycle, and the processes need cell motility and its control by actin cytoskeletal reorganization. This study investigated actin depolymerizing factor (ADF)/cofilin (Cfl) family proteins, which are important molecules in actin cytoskeletal reorganization, in Entamoeba invadens in relation to the encystation and excystation. Axenic culture systems were used to induce encystation and excystation. A homology search of the E. invadens genome database and molecular cloning identified three ADF/Cfl family proteins of the parasite (named for short as EiCfl-1, EiCfl-2, and EiCfl-3). This is different from other Entamoeba species, i.e. Entamoeba histolytica and Entamoeba dispar, each of which has only one ADF/Cfl family protein. These ADF/Cfl of E. invadens do not have Ser3 (serine locates third from first methionine), similar to E. histolytica, E. dispar, Saccharomyces cerevisiae and Schizosaccharomyces pombe, although the activity of ADF/Cfl is negatively regulated by phosphorylation of the Ser3 in metazoans. Phylogenetic analysis revealed that Entamoeba Cfl formed a distinctive clade that is separate from other organisms, and the branches of the tree were separated in two consistent with the presence and absence of Ser3. Rabbit anti-EiCfl-2 serum reacted with all recombinant EiCfls and EiCfl in lysates of cysts, trophozoites and metacystic amoebae. Immunofluorescence staining with this antiserum showed co-localization of EiCfl with actin beneath the cell membrane through the life stages. Both proteins proved to be rich in pseudopodia of trophozoites and metacystic amoebae. Real-time RT-PCR showed that mRNAs of EiCfl-2 and actins were highly expressed, but there were few mRNA of EiCfl-1 and EiCfl-3. Remarkably decreased mRNA levels were observed in EiCfl-2 and actins during encystation. All three EiCfls and actins became transcribed after the induction of excystation. The mRNAs of only EiCfl-1 and EiCfl-3 increased remarkably when the excystation was induced in the presence of cytochalasin D. These findings demonstrate that EiCfl-2 and actins co-localize beneath the cell membrane in trophozoites and cysts as well as metacystic amoebae being rich in pseudopodia, that EiCfl-1 and EiCfl-3 are expressed only after the induction of excystation, and that enhanced excystation by cytochalasin D is associated with high expression of EiCfl-1 and EiCfl-3.

Different structure and mRNA expression of Entamoeba invadens chitinases in the encystation and excystation.

Entamoeba histolytica forms chitin-walled cysts during encystation process, where formation of the cyst wall needs not only chitin synthase but also chitinase. During excystation, quadruplet amoebae emerge from the chitin-walled cysts by dissolving the wall, so that chitinase may be necessary for excystation process as well. There is, however, no report on chitinase expression during excystation. In this study, we used Entamoeba invadens, a reptilian amoeba, as a model for encystation and excystation of E. histolytica, and studied chitinase mRNA expression in those processes. Although expression of three E. invadens chitinases designated EiChit1, EiChit2, and EiChit3 during encystation has been reported, we identified another enzyme named as EiChit4 in the E. invadens genome database. Therefore, we investigated the primary structure and mRNA expression of these four chitinases of Ei in the excystation as well as the encystation by real-time reverse transcription polymerase chain reaction (RT-PCR). Like EiChit1, EiChit4 had an 8 × Cys chitin-binding domain (CBD) and a hydrophilic spacer between the CBD and catalytic domain, and was also closer to EiChit1 than EiChit2 and EiChit3 in the phylogenetic tree. During encystation, the expression of all four chitinases increased in the early phase; the increase in EiChit1 and EiChit4 was much higher than in EiChit2 and EiChit3. Then, the expression of all four chitinases sharply decreased in the later phase. In cysts, EiChit1 was most abundantly expressed and EiChit4 was at a lower level, while the expressions of EiChit2 and EiChit3 were virtually absent. Following the induction of excystation, mRNA levels of EiChit1 and EiChit4 in cysts 5 h after induction were significantly lower than those in cysts before induction, while those of EiChit2 and EiChit3 were remarkably higher than before induction. The mRNAs of only EiChit2 and EiChit3 remarkably increased when the excystation was induced in the presence of cytochalasin D. These data demonstrate different structures and expressions of four chitinases in the differentiation of E. invadens.

Involvement of serine proteases in the excystation and metacystic development of Entamoeba invadens.

Although the functions of cysteine proteases involved in the pathogenicity and differentiation of Entamoeba histolytica have been demonstrated, little is known about the functions of serine proteases. We examined the involvement of serine proteases in amoebic excystation and metacystic development using inhibitors specific for serine proteases. Entamoeba invadens IP-1 strain was used as the model of excystation and metacystic development of E. histolytica. Four serine protease inhibitors, phenylmethanesulfonyl fluoride (PMSF), 4-(2-aminoethyl) bezensulfonylfluoride hydrochloride, 3, 4-dichloroisocoumarin, and N-tosyl-phe-chloromethylketone, decreased the number of metacystic amoebae in a dose-dependent manner, without showing cytotoxicity to cysts. PMSF inhibited not only the increase but also the development of metacystic amoebae as determined by the change of nucleus number from four- to one-nucleate amoebae. The protease activity in cyst lysates was also inhibited by PMSF and the band of protease on gelatin sodium dodecyl sulfate polyacrylamide gel electrophoresis was weaker than controls when treated with PMSF. Three serine protease families, S28 (three types), S9 (two), and S26 (one) were retrieved from the database of E. invadens. Phylogenetic analysis revealed that amebic enzymes from the serine protease families formed different clades from those from other organisms. The expression levels of these serine proteases in cysts 5 h after the induction of excystation as assessed by real-time reverse transcriptase polymerase chain reaction (RT-PCR) were higher than those observed prior to induction assayed by real-time RT-PCR; the increase in one type of S9 (named S9-3) expression was the highest. The expression of S9 enzymes also increased from cysts to trophozoites higher than the other family serine proteases. Thus, the results show that Entamoeba uses their serine proteases in the excystation and metacystic development, which leads to successful infection.

Differences in protein profiles of the isolates of Entamoeba histolytica and E. dispar by surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF MS) ProteinChip assays.

Surface-enhanced laser desorption ionization time of flight mass spectrometry (SELDI-TOF MS) ProteinChip assays with weak cationic exchange chips were used for protein profiling of different isolates of Entamoeba histolytica and E. dispar. When SELDI-TOF MS spectra of cell lysates from E. histolytica strain HM-1:IMSS were compared with those from four other laboratory strains (200:NIH, HK-9, DKB, and SAW755CR) grown under the same culture conditions, different peak patterns of SELDI-TOF MS were observed among these strains, independent of their zymodeme types. Similarly, five Japanese isolates of E. histolytica grown under the same culture conditions revealed different peak patterns among themselves. The SELDI-TOF MS spectra of cell lysates from two isolates of E. dispar strain AS16IR and CYNO 09:TPC showed the presence of peaks specific for E. dispar isolates and the absence of peaks common to E. histolytica isolates. This is not only the first use of SELDI-TOF MS ProteinChip technology for protein profiling of different strains of Entamoeba but also the use for parasitic protozoa. The SELDI-TOF MS spectra show a realistic view of proteins with a biological status of E. histolytica and E. dispar isolates, contributing to show their phenotypic differences of proteins and provide a unique means of distinguishing them.

Metastasis in the mandibular condyle: a case report.

BACKGROUND: Most bone metastases are observed in the trunk of the body. Metastasis in the mandibular condyle is rare. In many case reports, temporary common temporomandibular joint disorder-like symptoms can be a sign of relapse and metastasis. CASE PRESENTATION: We report a rare case of breast carcinoma metastatic to the left mandibular condyle in a 55-year-old Japanese woman, who visited our department for a dental check-up prior to chemotherapy. She had almost no symptoms, but radiographs suggested the existence of metastasis. CONCLUSIONS: In many case reports, patients had some symptoms. In this case report, our patient had slight symptoms, but we were able to confirm the metastasis from the symptoms and panoramic dental radiograph. When patients complain about discomfort of the temporomandibular joint, we need to consider the possibility of metastasis and notice changes on the panoramic dental radiograph.

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.

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.

[Gnathostomiasis caused by Gnathostoma spinigerum etiologically diagnosed upon extraction of the worm from the skin].

We report a patient with gnathostomiasis in whom a specific diagnosis of Gnathostoma spinigerum infestation was made morphologically upon removal of the worm. A 47-Year-old Japanese male on a business trip to Vietnam ate fried frog with a Vietnamese friend in January 2002, the friend was diagnosed with gnathostomiasis in June 2002. The patient noted swelling of the right leg with migration to the right arm, prompting him to our hospital in February 2003. Hematologic examination showed eosinophilia, and specific anti-gnathostome antibody was detected by a dot enzyme-linked immunosorbent assay (Dot ELISA) in the serum. He was diagnosed as gnathostomiasis, and was given albendazole 400 mg b.i.d. On day 11 of therapy the patient removed a larval worm from the right palmar lesion by pinching with his nails. The worm was identified as G. spinigerum based on morphologic characteristics including number of hooklets on its head-bulb. When gnathostomiasis is suspected, albendazole should be administered before incision of the skin lesion.

A case of cutaneous myiasis due to Dermatobia hominis in Japan.

We report the 34th imported case of cutaneous myiasis caused by Dermatobia hominis in Japan, which is not a habitat of the fly. A 41-year-old Japanese man noticed an insect-sting-like papule on his left upper back during his stay in Ecuador in March 2004. After his return home, the lesion gradually increased to become a red subcutaneous nodule with a central pore from which serosanguineous fluid drained. Because antimicrobial treatment under a diagnosis of inflammatory atheroma was ineffective, the lesion was incised and a 3rd instar larva of D. hominis was then found and removed. We checked the literature on D. hominis myiasis reported from Japan, and noted the fact, which nobody had previously pointed out, that in Japan only one case of D. hominis myiasis had been diagnosed correctly before a larva was found, and most of the cases were misdiagnosed and inappropriately treated, including 11 cases given unnecessary resection of the nodules. Doctors in Japan should be aware of myiasis so that patients are neither anxious about the disease nor suffer pain, and doctors avoid performing unnecessary resections of the lesions.

Long-term cigarette smoking increases the prevalence of carotid artery calcification seen on panoramic dental radiographs in male patients.

Panoramic dental radiographs are commonly used in general dentistry and oral and maxillofacial surgery to examine upper and lower teeth, maxilla, mandible and the surroundings simultaneously. Carotid artery calcification, a specific indicator of atherosclerotic change of the carotid arteries, can be seen on the radiographs. Many studies have suggested that cigarette smoking is a risk factor of atherosclerotic change as well as cerebral infarction. We hypothesized that smoking could increase the prevalence of carotid artery calcification, and compared the radiographs of smokers and non-smokers aged 50 years and over: 146 male smokers, 165 male non-smokers, 42 female smokers and 422 female non-smokers. This is the first study to focus on carotid artery calcification seen on panoramic dental radiographs to show the connection between smoking and atherosclerotic change. In male patients, carotid artery calcification was seen in 18 (14.1%) of the smokers, and in 8 (4.8%) of the non-smokers, which clearly shows that male patients aged 50 years old or over are more likely to develop carotid artery calcification if they smoke. However, there is no significant difference between female smokers and female non-smokers in the same age group. Dentists are in a good position to find carotid artery calcification on radiographs. When this is found on a radiograph, the patient should be advised to stop smoking and be referred to a physician for further tests. Clinicians should be aware that this radiographic finding indicates the presence of atherosclerotic change of the carotid arteries.

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.

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.

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.

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.