Identification of asymptomatic Entamoeba histolytica infection by a serological screening test: A cross-sectional study of an HIV-negative men who have sex with men cohort in Japan

BackgroundAmebiasis, caused by Entamoeba histolytica, is spreading in developing countries and in many developed countries as a sexually transmitted infection. Here, we evaluated the efficacy of serological screening to identify asymptomatic E. histolytica infection as a potential epidemiological control measure to limit its spread.Methodology/Principal findingsThis cross-sectional study was carried out between January and March 2021 in an HIV-negative men who have sex with men (MSM) cohort at the National Center for Global Health and Medicine. Serological screening was performed using a commercially available ELISA kit. For seropositive individuals, we performed stool polymerase chain reaction (PCR) to determine current E. histolytica infection. We performed E. histolytica serological screening of 312 participants. None had a history of E. histolytica infection prior to the study. The overall E. histolytica seropositivity was 6.7% (21/312), which was similar to that found by the rapid plasma reagin test (17/312). We identified current infection in 8 of 20 seropositive participants (40.0%) by stool PCR.Conclusions/SignificanceOur serological screening approach constitutes a potentially practical epidemiological strategy. Active epidemiological surveys, in combination with an effective screening strategy for asymptomatically infected individuals, should be applied to help reduce sexually transmitted E. histolytica infections.     

Involvement of rho p21 and its inhibitory GDP/GTP exchange protein (rho GDI) in cell motility.

Evidence is accumulating that rho p21, a ras p21-related small GTP-binding protein (G protein), regulates the actomyosin system. The actomyosin system is known to be essential for cell motility. In the present study, we examined the action of rho p21, its inhibitory GDP/GTP exchange protein (named rho GDI), its stimulatory GDP/GTP exchange protein (named smg GDS), and Clostridium botulinum ADP-ribosyltransferase C3, known to selectively ADP-ribosylate rho p21 and to impair its function, in cell motility (chemokinesis) of Swiss 3T3 cells. We quantitated the capacity of cell motility by measuring cell tracks by phagokinesis. Microinjection of the GTP gamma S-bound active form of rhoA p21 or smg GDS into Swiss 3T3 cells did not affect cell motility, but microinjection of rho GDI into the cells did inhibit cell motility. This rho GDI action was prevented by comicroinjection of rho GDI with the GTP gamma S-bound form of rhoA p21 but not with the same form of rhoA p21 lacking the C-terminal three amino acids which was not posttranslationally modified with lipids. The rho GDI action was not prevented by Ki-rasVal-12 p21 or any of the GTP gamma S-bound form of other small GTP-binding proteins including rac1 p21, G25K, and smg p21B. Among these small G proteins, rhoA p21, rac1 p21, and G25K are known to be substrates for rho GDI. The rho GDI action was not prevented by comicroinjection of rho GDI with smg GDS. Microinjection of C3 into Swiss 3T3 cells also inhibited cell motility. These results indicate that the rho GDI-rho p21 system regulates cell motility, presumably through the actomyosin system.     

SGIP1alpha is an endocytic protein that directly interacts with phospholipids and Eps15

SGIP1 has been shown to be an endophilin-interacting protein that regulates energy balance, but its function is not fully understood. Here, we identified its splicing variant of SGIP1 and named it SGIP1alpha. SGIP1alpha bound to phosphatidylserine and phosphoinositides and deformed the plasma membrane and liposomes into narrow tubules, suggesting the involvement in vesicle formation during endocytosis. SGIP1alpha furthermore bound to Eps15, an important adaptor protein of clathrin-mediated endocytic machinery. SGIP1alpha was colocalized with Eps15 and the AP-2 complex. Upon epidermal growth factor (EGF) stimulation, SGIP1alpha was colocalized with EGF at the plasma membrane, indicating the localization of SGIP1alpha at clathrin-coated pits/vesicles. SGIP1alpha overexpression reduced transferrin and EGF endocytosis. SGIP1alpha knockdown reduced transferrin endocytosis but not EGF endocytosis; this difference may be due to the presence of redundant pathways in EGF endocytosis. These results suggest that SGIP1alpha plays an essential role in clathrin-mediated endocytosis by interacting with phospholipids and Eps15.     

Introduction of a non-native disulfide bridge to human lysozyme by cysteine scanning mutagenesis

To examine whether the disulfide bridge between residues 65 and 81 can be replaced by a non-native disulfide bridge in the mutant h-lysozyme C77/95A and whether the formation of such a new disulfide bridge affects the folding of the protein, cysteine scanning mutagenesis has been performed within two discontinuous segments (residues 61-67 for the mutant C65/77/95A, and 74-84 for the mutant C77/81/95A). The position of the Cys residue at 65 or 81 was continuously shifted by site-directed mutagenesis. Of the mutants, only substitution of Cys for Trp64 allowed the secretion of mutant h-lysozyme(W64C) into the medium in a sufficient amount for analysis. After the purification, the mutant enzyme was obtained as two components (W64C-A and W64C-B). The only difference between A and B was that A had a peptide bond cleaved between Ala77 and His78. A non-native disulfide bridge between residues 64-81 was found in both components. Little difference was observed in CD spectra among wild-type and mutant enzymes. It is likely that the tertiary structure of the W64C mutant might be distorted at the location, because the directions of amino acid side chains at positions of 64 and 81 are shown to be opposite to each other in wild-type h-lysozyme by X-ray crystallographic analysis.     

Effects of population density, sex morph, and tree size on reproduction in a heterodichogamous maple, Acer mono, in a temperate forest of Japan

The effects of local population density, sex morph [protogynous (PG) or protandrous (PA)], and individual tree size on the demographic processes of seed production were investigated in a heterodichogamous maple, Acer mono Maxim. var. Marmoratum (Nichols.) Hara f. dissectum, in a temperate forest of Japan. As the distance from conspecific reproductive adults increased, the percentage of immature seed fall and empty seeds increased significantly, indicating higher pollination success along with local population density. Although the difference was not distinct, pollination success was affected by the local population density of the reciprocal sex morph rather than that of both sex morphs. The trees at higher local population density sites suffered higher seed mortality due to predation and decay, and tended to produce smaller seeds. Thus, the impacts of local population density operated both positively and negatively on reproduction. As a factor of individual traits, tree size scarcely affected any demographic processes. On the other hand, sex morph did affect pollination success. Trees of PG type had lower immature seed fall than those of PA type, suggesting that the former has higher efficiency of pollen acceptance than the latter. The results on seed demography presented here partly support previous suggestions that heterodichogamous plants exhibit reciprocal cross-pollination and gender specialization as reproductive traits.     

The bacteriophage lambda int gene product. A filter assay for genetic recombination, purification of int, and specific binding to DNA.

Bacteriophage lambda int gene is required for the integration of viral DNA into the chromosome of Escherichia coli. We have extensively purified the product of the int gene (Int) from a lysogen of E. coli that constitutively expresses this gene. Int was assayed by its ability to promote integrative recombination of supertwisted substrate DNA in vitro using a new method based on filter trapping of a recombinant product DNA. In order to catalyze integrative recombination, Int must be supplemented by other factors that can be extracted from bacterial host cells. By itself, purified Int does not demonstrate detectable endonuclease, exonuclease, or nicking-closing activities. However, Int does make stable complexes with double-stranded lambda-DNA containing an attachment site, the region at which recombination takes place. No stable complexes are observed between Int and lambda-DNA without an attachment site or between Int and DNA containing the bacterial site of integration. Int, therefore, appears to be a specificity element that relies on additional factor(s) to provide or activate the catalytic functions required for recombination.     

Molecular cloning and characterization of a novel type of regulatory protein (GDI) for smg p25A, a ras p21-like GTP-binding protein.

We recently purified to near homogeneity a novel type of regulatory protein for smg p25A, a ras p21-like GTP-binding protein, from bovine brain cytosol. This regulatory protein, named smg p25A GDP dissociation inhibitor (GDI), regulates the GDP-GTP exchange reaction of smg p25A by inhibiting dissociation of GDP from and subsequent binding of GTP to it. In the present studies, we isolated and sequenced the cDNA of smg p25A GDI from a bovine brain cDNA library by using an oligonucleotide probe designed from the partial amino acid sequence of purified smg p25A GDI. The cDNA has an open reading frame that encodes a protein of 447 amino acids with a calculated Mr of 50,565. This Mr is similar to those of the purified smg p25A GDI estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sucrose density gradient ultracentrifugation, which are about 54,000 and 65,000, respectively. The isolated cDNA is expressed in Escherichia coli, and the encoded protein exhibits GDI activity. smg p25A GDI is hydrophilic overall, except for one hydrophobic region near the N terminus. smg p25A GDI shares low amino acid sequence homology with the Saccharomyces cerevisiae CDC25-encoded protein, which has been suggested to serve as a factor that regulates the GDP-GTP exchange reaction of the yeast RAS2-encoded protein, but not with the beta gamma subunits of GTP-binding proteins having an alpha beta gamma subunit structure, such as Gs and Gi. The smg p25A GDI mRNA was present in various tissues, including not only tissues in which smg p25A was detectable but also tissues in which it was not detectable. This fact has raised the possibility that smg p25A GDI interacts with another G protein in tissues in which smg p25A is absent.     

Characterization of the cells in the repair tissue of full-thickness articular cartilage defects

 It is well established that a full-thickness articular cartilage defect is repaired with a fibrocartilaginous tissue, cells of which are derived from undifferentiated mesenchymal stem cells in the bone marrow. To characterize the repair cells biochemically, full-thickness defects were created in rabbit knee joints and the repair tissues taken at 3, 6, and 12 weeks after surgery. The repair cells were cultured and examined biochemically to investigate the effects of four exogenous growth factors with regard to the metabolism of type II collagen and proteoglycans. A significant increase of carboxy-terminal type II procollagen peptide production was observed in the conditional medium of the repair cells, especially taken at 6 weeks after surgery, in the presence of each growth factor. Glycosaminoglycan content was also increased and proteoglycan synthesis stimulated. The repair cells taken at the early stage of the repair process could originally have more activity of type II collagen synthesis, and the growth factors used could enhance the differentiation of the repair cells in vitro. Accepted: 3 November 1997     

Establishment of ex vivo systems to identify compounds acting on innate immune responses and to determine their target molecules using transgenic Drosophila

Innate immunity is an evolutionarily conserved self-defense mechanism against microbial infections. In Drosophila, induction of antimicrobial peptides is a major immune response that is regulated by two distinct signaling pathways called the IMD pathway and the Toll pathway, similar to the tumor necrosis factor-alpha signaling and Toll-like receptor/interleukin-1 signaling pathways, respectively, in mammals. In mammals, innate immunity interacts with adaptive immunity and has a key role in the regulated immune response. Therefore, innate immunity is a pharmaceutical target for the development of immune regulators. Previously, based on the striking conservation between the mechanisms that regulate Drosophila immunity and human innate immunity, we established an ex vivo culture in which compounds acting on innate immunity can be evaluated using a reporter gene that reflects activation of the IMD pathway [Yajima et al. [Yajima, M., Takada, M., Takahashi, N., Kikuchi, H., Natori, S., Oshima, Y., Kurata, S., 2003. A newly established in vitro culture using transgenic Drosophila reveals functional coupling between the phospholipase A2-generated fatty acid cascade and lipopolysaccharide-dependent activation of the immune deficiency (imd) pathway in insect immunity. The Biochemical Journal 371(Pt 1), 205-210] Biochem J 371, 205-210]. Here, we combined the ex vivo culture with a reporter gene that reflects the heat shock response and demonstrated that the resulting systems are useful for screening compounds that act specifically on innate immunity, including mammalian innate immune responses. Identification of target molecules is essential for the development of more potent medicines with fewer side effects. In this study, we also established ex vivo systems capable of identifying target molecules of the identified compounds using targeted activation of the IMD pathway.