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.

A recombinant matrix metalloproteinase protein from Gnathostoma spinigerum for serodiagnosis of neurognathostomiasis.

Neurognathostomiasis is a severe form of human gnathostomiasis which can lead to disease and death. Diagnosis of neurognathostomiasis is made presumptively by using clinical manifestations. Immunoblotting, which recognizes antigenic components of molecular mass 21 kDa and 24 kDa in larval extracts of Gnathostoma spinigerum (Gs 21/24), has high sensitivity and specificity for diagnosis of neurognathostomiasis. However, only very small amounts of the Gs 21/24 antigens can be prepared from parasites harvested from natural or experimental animals. To overcome this problem, we recently produced a recombinant matrix metalloproteinase (rMMP) protein from G. spinigerum. In this study, we evaluated this rMMP alongside the Gs 21/24 antigens for serodiagnosis of human neurognathostomiasis. We studied sera from 40 patients from Srinagarind Hospital, Khon Kaen University, Thailand, with clinical criteria consistent with those of neurognathostomiasis, and sera from 30 healthy control adults from Thailand. All sera were tested for specific IgG antibodies against both G. spinigerum crude larval extract and rMMP protein using immunoblot analysis. The sensitivity and specificity for both antigenic preparations were all 100%. These results show that G. spinigerum rMMP protein can be used as an alternative diagnostic antigen, in place of larval extract, for serodiagnosis of neurognathostomiasis.

A Recombinant Matrix Metalloproteinase Protein from Gnathostoma spinigerum for Serodiagnosis of Neurognathostomiasis

Neurognathostomiasis is a severe form of human gnathostomiasis which can lead to disease and death. Diagnosis of neurognathostomiasis is made presumptively by using clinical manifestations. Immunoblotting, which recognizes antigenic components of molecular mass 21 kDa and 24 kDa in larval extracts of Gnathostoma spinigerum (Gs 21/24), has high sensitivity and specificity for diagnosis of neurognathostomiasis. However, only very small amounts of the Gs 21/24 antigens can be prepared from parasites harvested from natural or experimental animals. To overcome this problem, we recently produced a recombinant matrix metalloproteinase (rMMP) protein from G. spinigerum. In this study, we evaluated this rMMP alongside the Gs 21/24 antigens for serodiagnosis of human neurognathostomiasis. We studied sera from 40 patients from Srinagarind Hospital, Khon Kaen University, Thailand, with clinical criteria consistent with those of neurognathostomiasis, and sera from 30 healthy control adults from Thailand. All sera were tested for specific IgG antibodies against both G. spinigerum crude larval extract and rMMP protein using immunoblot analysis. The sensitivity and specificity for both antigenic preparations were all 100%. These results show that G. spinigerum rMMP protein can be used as an alternative diagnostic antigen, in place of larval extract, for serodiagnosis of neurognathostomiasis.

Identification and characterization of a cathepsin L-like cysteine protease from Gnathostoma spinigerum.

Gnathostoma spinigerum is a causative agent of human gnathostomiasis, a common parasitic disease involving skin and visceral organs, especially the central nervous system. In this study, we identified a cDNA encoding a cathepsin L-like cysteine protease (GsCL1) from the lambdaZAP cDNA library of G. spinigerum advanced third-stage larva (aL3) and characterized the biochemical properties of the recombinant enzyme. The cloned cDNA of 1484bp encoded 398 amino acids which contained a typical signal peptide sequence (23 amino acids), a pro-domain (156 amino acids), and a mature domain (219 amino acids) with an approximate molecular weight of 24kDa. The deduced amino acid sequence of GsCL1 gene showed 53-64% identity to cathepsin L proteases of various organisms including a cathepsin L family member (cpl-1) of Caenorhabditis elegans. Recombinant proGsCL1 expressed in Pichia pastoris showed typical biochemical characteristics of cysteine proteases. The expressed enzyme displayed optimal protease activity toward Z-Phe-Arg-AMC substrate at pH 6.0 but not toward Z-Arg-Arg-AMC. The activity was sensitive to cysteine protease inhibitors E-64 and K11777. The preference for large hydrophilic and aromatic residues in the P2 position (I, L, F, W, U, V) was typical of cathepsin L proteases. Mouse anti-GST-proGsCL1 serum showed reactivity with 35-, 38- and 45-kDa proteins in the aL3 extracts. These proteins were shown to localize inside the intestinal cells of aL3.

Activities of antioxidant enzymes and cytochrome c oxidase in liver of Arctic and temperate teleosts.

Enhanced antioxidant status in polar fishes may occur due to high dissolved oxygen levels and membranes rich in peroxidation-sensitive polyunsaturated fatty acids. To evaluate the importance of antioxidant enzymes in polar fishes, activities of catalase (CAT), superoxide dismutase (SOD), and glutathione reductase (GR), as well as the aerobic enzyme cytochrome c oxidase (CCO), were measured at 6 degrees C and 1 degrees C in livers of confamilial Arctic and temperate teleosts: the Arctic fourhorn sculpin Triglopsis quadricornis (Cottidae) and saddled eelpout Lycodes mucosus (Zoarcidae) vs. the temperate longhorn sculpin (Myoxocephalus octodecimspinosus) (Cottidae) and ocean pout (Zoarces americanus) (Zoarcidae), respectively. At both assay temperatures, CAT activities were substantially lower in both Arctic species, SOD was similar in the cottids but lower in the Arctic zoarcid, and GR was similar in temperate and Arctic fishes. Activities at respective habitat temperatures were always significantly lower in the Arctic fishes. The lower antioxidant enzyme activities in the Arctic fishes cannot be attributed to lower aerobic status because CCO activity was similar or higher in the Arctic fishes; significant negative relationships were found between CCO and CAT and GR (but not SOD) when all species were combined, indicating that a higher apparent aerobic status does not necessarily coincide with higher antioxidant enzyme activities. Antioxidant enzyme activities may not be enhanced as part of cold adaptation in Arctic fishes, at least in the liver.

Gnathostoma binucleatum: excretion-secretion antigen analysis obtained from advanced third-stage larvae in in vitro culture.

The paper describes an introductory characterization of antigenic stimulation of excretion-secretion products (ESP) of Gnathostoma binucleatum advanced third-stage larvae cultured in vitro and proteinases present in this products. Excretory and secretory proteins were obtained after 10 larvae were maintained in 5% CO(2) RPMI medium. The supernatant was collected each week for two months. The proteins were dialyzed, concentrated, and separated in 10% SDS-PAGE gels under reducing conditions and transferred to nitrocellulose paper for immunoblot analyses. G. binucleatum immunized mice serum was used to determine protein antigenicity. Four proteins of 40, 80, 120, and 208 kDa persisted for two months and three proteins, 80, 120, and 208 kDa were recognized for antibodies of mice. In SDS-PAGE gelatin substrate gels ESP resolved as two proteins with molecular weight of 80 and 208 kDa that were sensitive to a metalloproteinase inhibitor, and thus it may be inferred that they might be used for diagnosis of gnathostomiasis.

Molecular cloning of a gene encoding matrix metalloproteinase-like protein from Gnathostoma spinigerum.

The advanced third-stage larvae (aL3) of Gnathostoma spinigerum contain a 24 kDa glycoprotein with diagnostic potential. Immunoscreening with the monoclonal antibody to the 24-kDa protein (mAb GN6/ 24) has identified a cDNA clone with an insert of 932 base pairs (bp). The insert contains a full-length gene of 732 bp encoding a protein that is 33-39% similar to matrix metalloproteinases (MMPs) of Caenorhabditis elegans and several lower and higher vertebrates. The MMP-like protein of G. spinigerum possesses the catalytic domain, but lacks the propeptide and hemopexin-like domains found in other MMPs. A signal peptide of 23 amino acids at its amino terminus indicates that it is a secretory protein, which is confirmed by Western blot analysis showing the presence of the 24 kDa protein in the excretory-secretory products of aL3.