PML and PML-like exonucleases restrict retrotransposons in jawed vertebrates.

We have uncovered a role for the promyelocytic leukemia (PML) gene and novel PML-like DEDDh exonucleases in the maintenance of genome stability through the restriction of LINE-1 (L1) retrotransposition in jawed vertebrates. Although the mammalian PML protein forms nuclear bodies, we found that the spotted gar PML ortholog and related proteins in fish function as cytoplasmic DEDDh exonucleases. In contrast, PML proteins from amniote species localized both to the cytoplasm and formed nuclear bodies. We also identified the PML-like exon 9 (Plex9) genes in teleost fishes that encode exonucleases. Plex9 proteins resemble TREX1 but are unique from the TREX family and share homology to gar PML. We also characterized the molecular evolution of TREX1 and the first non-mammalian TREX1 homologs in axolotl. In an example of convergent evolution and akin to TREX1, gar PML and zebrafish Plex9 proteins suppressed L1 retrotransposition and could complement TREX1 knockout in mammalian cells. Following export to the cytoplasm, the human PML-I isoform also restricted L1 through its conserved C-terminus by enhancing ORF1p degradation through the ubiquitin-proteasome system. Thus, PML first emerged as a cytoplasmic suppressor of retroelements, and this function is retained in amniotes despite its new role in the assembly of nuclear bodies.

Molecular cloning and characterization of serine protease inhibitor from food-borne nematode, Gnathostoma spinigerum.

Gnathostoma spinigerum is a causative agent of human gnathostomiasis and infects people residing in endemic areas as well as travelers. Cutaneous and visceral larval migrants cause clinical manifestations, resulting in severe morbidity and mortality. To survive in hosts, these parasites have evolved various immune evasion mechanisms, including the release of regulatory molecules. Serine protease inhibitors (serpins) that are present in many parasitic helminths are proteins suspected of suppressing host serine protease-related digestion and immune responses. In this study, the serpin secreted by G. spinigerum (GsSerp) was characterized using bioinformatics and molecular biology techniques. The bioinformatics revealed that GsSerp contains 9 helices, 3 ß-sheets, and a reactive central loop, which are conserved structures of the serpin superfamily. Recombinant GsSerp (rGsSerp) was expressed in E. coli (molecular weight, 39 kDa) and could inhibit chymotrypsin. Mouse polyclonal antibody against GsSerp could detect the native GsSerp in crude worm antigen but not the excretory-secretory product (ES) of infective-stage larva (aL3Gs). Moreover, the expression of GsSerp in the aL3Gs tissue was located in the hemolymph and intestinal tissue, indicating its role in parasite homeostasis. Our findings may help develop effective strategies for preventing and controlling gnathostomiasis.

Excretory-secretory product of third-stage Gnathostoma spinigerum larvae induces apoptosis in human peripheral blood mononuclear cells.

Human gnathostomiasis caused by third-stage Gnathostoma spinigerum larvae (G. spinigerum L3) is an important zoonotic disease in tropical areas of the world. The excretory-secretory products (ES) that are excreted by infective larva play a significant role in host immune evasion and tissue destruction. To investigate the poorly understood mechanisms of G. spinigerum L3 pathogenesis, we focused on the potential effect of ES on inducing apoptosis in human immune cells by using human peripheral blood mononuclear cells (PBMCs) as a model. Early and late apoptosis of PBMCs were assessed following the exposure of these cells to G. spinigerum L3 ES (0.1, 0.5, and 1.0 µg/ml) for 6-48 h. The apoptotic cells were identified by flow cytometric staining of PBMC with FITC-annexin V and propidium iodide. The expression of regulatory genes related to apoptosis mechanisms in ES-treated PBMCs was investigated using a Human Apoptosis RT2 Profiler™ PCR Array. The results showed significant levels of early phase apoptosis at 18 h and of late phase apoptosis at 24 h. We speculate that this apoptosis in PBMCs occurs via the extrinsic pathway. Apoptosis in the ES-induced PBMCs was observed as quickly as 90 min after exposure, and the highest effect was observed at 18-24 h. Furthermore, ES can trigger apoptosis lasting for 48 h. Our findings expand the understanding of one of the mechanisms involved, immune-evasive strategy mechanism used by G. spinigerum larvae during human gnathostomiasis.

Gnathostoma spinigerum: molecular cloning, expression and characterization of the cyclophilin protein.

In this study, a cDNA encoding cyclophilin (CyP) of Gnathostoma spinigerum was cloned into a prokaryotic expression vector and expressed in Escherichia coli. The predicted molecular mass of the putative protein was 18.6kDa, and the deduced amino acid sequence had 86, 84.8, 81.3 and 77.2% identity with the CyP of Dirofilaria immitis, Brugia malayi, Onchocerca volvulus and Caenorhabditis elegans, respectively. A prediction of linear B-cell epitopes with high hydrophilicity and immunoblotting results indicated that the recombinant CyP has antigenicity to humans. The recombinant CyP protein reacted with human gnathostomiasis sera but not with other parasitosis or healthy control sera, suggesting that it might be useful for the serodiagnosis of human gnathostomiasis.

Protein profile analysis from advanced third-stage larvae (AdvL3) and adult worms of Gnathostoma binucleatum (Nematoda: Spirurida).

Proteins from crude extracts of advanced third-stage larvae and adult Gnathostoma binucleatum nematode worms showed protein profiles in SDS-PAGE analysis similar to Echinococcus granulosus, Trichinella spiralis, Dipylidium caninum, Ancylostoma caninum, Ascaris lumbricoides and Toxocara canis. The immunoblot analysis of the human serum infected or suspected to be infected with G. binucleatum using the total larvae extract recognized the 40, 60, 80 and 115 kDa proteins and using the total adult worm extract recognized only the 80 and 115 kDa proteins. However, the 115 kDa protein showed cross-reactions with A. caninum, A. lumbricoides, T. canis and D. caninum with human serum positive to gnathostomosis, while the 40 kDa protein was only recognized with the G. binucleatum total larvae extract. The results obtained suggest that the use of antigens from the advanced third-stage larvae of the parasite were best recognized for immunodiagnosis of gnathostomosis.

Trends in the evolution of the prodynorphin gene in teleosts: cloning of eel and tilapia prodynorphin cDNAs.

The detection of the prodynorphin gene in anuran amphibians and lungfishes may indicate that this gene arose as a result of the duplication of the proenkephalin gene early during the divergence of the Sarcopterygii, or that this gene may predate the divergence of the ray-finned fish and the lobe-finned fish. The cloning of prodynorphin-related genes from the pufferfish and zebrafish supports the latter hypothesis. This study analyzes trends in the radiation of the prodynorphin gene in teleosts. Prodynorphin cDNAs were cloned from the brain of the eel Anguilla rostrata and the Nile tilapia, Oreochromis niloticus. These teleost prodynorphin sequences have distinct alpha-neoendorphin, dynorphin A, and dynorphin B sequences, and a novel opioid sequence, YGGFI. The relationship of these teleost prodynorphin sequences to other actinopterygian and sarcopterygian prodynorphin sequences will be discussed.

The origin of the parathyroid gland.

It has long been held that the parathyroid glands and parathyroid hormone evolved with the emergence of the tetrapods, reflecting a need for new controls on calcium homeostasis in terrestrial, rather than aquatic, environments. Developmentally, the parathyroid gland is derived from the pharyngeal pouch endoderm, and studies in mice have shown that its formation is under the control of a key regulatory gene, Gcm-2. We have used a phylogenetic analysis of Gcm-2 to probe the evolutionary origins of the parathyroid gland. We show that in chicks, as in mice, Gcm-2 is expressed in the pharyngeal pouches and the forming parathyroid gland. We find that Gcm-2 is present not only in tetrapods but also in teleosts and chondrichthyans, and that in these species, Gcm-2 is expressed within the pharyngeal pouches and internal gill buds that derive from them in zebrafish (Danio rerio), a teleost, and dogfish (Scyliorhinus canicula), a chondrichthyan. We further demonstrate that Gcm-2 is required for the formation of the internal gill buds in zebrafish. We also have identified parathyroid hormone 1/2-encoding genes in fish and show that these genes are expressed by the gills. We further show that the gills express the calcium-sensing receptor, which is used in tetrapods to monitor serum calcium levels. These results indicate that the tetrapod parathyroid gland and the gills of fish are evolutionarily related structures, and that the parathyroid likely came into being as a result of the transformation of the gills during tetrapod evolution.