Phylogeny of hymenolepidid cestodes (Cestoda: Cyclophyllidea) from mammalian hosts based on partial 28S rDNA, with focus on parasites from shrews.

The aims of the study are to enrich the partial 28S rDNA dataset for hymenolepidids by adding new sequences for species parasitic in the genera Sorex, Neomys and Crocidura (Soricidae) and to propose a new hypothesis for the relationships among mammalian hymenolepidids. New sequences were obtained for Coronacanthus integrus, C. magnihamatus, C. omissus, C. vassilevi, Ditestolepis diaphana, Lineolepis scutigera, Spasskylepis ovaluteri, Staphylocystis tiara, S. furcata, S. uncinata, Vaucherilepis trichophorus and Neoskrjabinolepis sp. The phylogenetic analysis (based on 56 taxa) confirmed the major clades identified by Haukisalmi et al. (Zool Scr 39:631-641, 2010) based on analysis of 31 species: Ditestolepis clade, Hymenolepis clade, Rodentolepis clade and Arostrilepis clade; however, the support was weak for the early divergent lineages of the tree and for the Arostrilepis clade. Novelties revealed include the molecular evidence for the monophyly of Coronacanthus, the non-monophyletic status of Staphylocystis and the polyphyly of Staphylocystoides. The analysis has confirmed the monophyly of Hymenolepis, the monophyly of hymenolepidids from glirids, the position of Pararodentolepis and Nomadolepis as sister taxa, the polyphyly of Rodentolepis, the position of Neoskrjabinolepis and Lineolepis as sister taxa, and the close relationship among the genera with the entire reduction of rostellar apparatus. Resolved monophyletic groups are supported by the structure of the rostellar apparatus. The diversification of the Ditestolepis clade is associated with soricids. The composition of the other major clades suggests multiple evolutionary events of host switching, including between different host orders. The life cycles of Coronacanthus and Vaucherilepis are recognised as secondarily aquatic as these taxa are nested in terrestrial groups.

The steroid derivative 6-aminocholestanol inhibits the DEAD-box helicase eIF4A (LieIF4A) from the Trypanosomatid parasite Leishmania by perturbing the RNA and ATP binding sites.

The antifungal agent 6-aminocholestanol targets the production of ergosterol, which is the principle sterol in many fungi and protozoans; ergosterol serves many of the same roles as cholesterol in animals. We found that it also is an effective inhibitor of the translation-initiation factor eIF4AI from mouse (eIF4AIMus) and the Trypanosomatid parasite Leishmania (LieIF4A). The eIF4A proteins belong to the DEAD-box family of RNA helicases, which are ATP-dependent RNA-binding proteins and RNA-dependent ATPases. DEAD-box proteins contain a commonly-shared core structure consisting of two linked domains with structural homology to that of recombinant protein A (RecA) and that contain conserved motifs that are involved in RNA and ATP binding, and in the enzymatic activity. The compound inhibits both the ATPase and helicase activities by perturbing ATP and RNA binding, and it is capable of binding other proteins containing nucleic acid-binding sites as well. We undertook kinetic analyses and found that the Leishmania LieIF4A protein binds 6-aminocholestanol with a higher apparent affinity than for ATP, although multiple binding sites were probably involved. Competition experiments with the individual RecA-like domains indicate that the primary binding sites are on RecA-like domain 1, and they include a cavity that we previously identified by molecular modeling of LieIF4A that involve conserved RNA-binding motifs. The compound affects the mammalian and Leishmania proteins differently, which indicates the binding sites and affinities are not the same. Thus, it is possible to develop drugs that target DEAD-box proteins from different organisms even when they are implicated in the same biological process.

Prospectively Isolated Tetraspanin+ Neoblasts Are Adult Pluripotent Stem Cells Underlying Planaria Regeneration.

Proliferating cells known as neoblasts include pluripotent stem cells (PSCs) that sustain tissue homeostasis and regeneration of lost body parts in planarians. However, the lack of markers to prospectively identify and isolate these adult PSCs has significantly hampered their characterization. We used single-cell RNA sequencing (scRNA-seq) and single-cell transplantation to address this long-standing issue. Large-scale scRNA-seq of sorted neoblasts unveiled a novel subtype of neoblast (Nb2) characterized by high levels of PIWI-1 mRNA and protein and marked by a conserved cell-surface protein-coding gene, tetraspanin 1 (tspan-1). tspan-1-positive cells survived sub-lethal irradiation, underwent clonal expansion to repopulate whole animals, and when purified with an anti-TSPAN-1 antibody, rescued the viability of lethally irradiated animals after single-cell transplantation. The first prospective isolation of an adult PSC bridges a conceptual dichotomy between functionally and molecularly defined neoblasts, shedding light on mechanisms governing in vivo pluripotency and a source of regeneration in animals. VIDEO ABSTRACT.

Molecular Cloning and Characterization of Ribosomal Protein RPS9 in Echinococcus granulosus.

Ribosomal protein S9 (RPS9) is an essential functional gene that participates in DNA repair and developmental regulations. A sequence homolog of RPS9 has been found to be upregulated in the protoscoleces (PSCs) of Echinococcus granulosus treated with artemisinin. However, E. granulosus RPS9 (EgRPS9) has not been identified before. In the present study, the 657-base pair (bp) cDNA encoding EgRPS9 was cloned. Amino acid sequence analysis showed that EgRPS9 was similar to the RSP9 proteins from Schistosoma japonicum (SjRPS9, 86%) and Schistosoma mansoni (SmRPS9, 79%). Phylogenetic tree analysis showed that EgRPS9, SmRPS9, and SjRPS9 were clustered together. We detected the EgRPS9 gene and protein expression in PSCs exposed to artesunate (AS) which displayed a dose-dependent reduction in PSC viability for 24 hr. The results showed that the EgRPS9 ratio of the 10-µM AS-treated ( P < 0.01) and 40-µM AS-treated ( P < 0.05) groups were increased from that of the control group. In addition, the level of reactive oxygen species (ROS) in the AS-treated groups increased in a dose-dependent manner compared to the level in the control group. In conclusion, the expression of EgRPS9 could be induced by ROS and might participate in the oxidative damage-based anti-parasite mechanism of AS treatment.

LIN41 Post-transcriptionally Silences mRNAs by Two Distinct and Position-Dependent Mechanisms.

The RNA-binding protein (RBP) LIN41, also known as LIN-41 or TRIM71, is a key regulator of animal development, but its physiological targets and molecular mechanism of action are largely elusive. Here we find that this RBP has two distinct mRNA-silencing activities. Using genome-wide ribosome profiling, RNA immunoprecipitation, and in vitro-binding experiments, we identify four mRNAs, each encoding a transcription factor or cofactor, as direct physiological targets of C. elegans LIN41. LIN41 silences three of these targets through their 3' UTRs, but it achieves isoform-specific silencing of one target, lin-29A, through its unique 5' UTR. Whereas the 3' UTR targets mab-10, mab-3, and dmd-3 undergo transcript degradation, lin-29A experiences translational repression. Through binding site transplantation experiments, we demonstrate that it is the location of the LIN41-binding site that specifies the silencing mechanism. Such position-dependent dual activity may, when studied more systematically, emerge as a feature shared by other RBPs.

Molecular cloning and characterization of glucose-6-phosphate dehydrogenase from Brugia malayi.

Glucose-6-phosphate dehydrogenase (G6PD), a regulatory enzyme of the pentose phosphate pathway from Brugia malayi, was cloned, expressed and biochemically characterized. The Km values for glucose-6-phosphate and nicotinamide adenine dinucleotide phosphate (NADP) were 0.25 and 0.014 mm respectively. The rBmG6PD exhibited an optimum pH of 8.5 and temperature, 40 °C. Adenosine 5' [γ-thio] triphosphate (ATP-γ-S), adenosine 5' [ß,γ-imido] triphosphate (ATP-ß,γ-NH), adenosine 5' [ß-thio] diphosphate (ADP-ß-S), Na+, K+, Li+ and Cu++ ions were found to be strong inhibitors of rBmG6PD. The rBmG6PD, a tetramer with subunit molecular weight of 75 kDa contains 0.02 mol of SH group per mol of monomer. Blocking the SH group with SH-inhibitors, led to activation of rBmG6PD activity by N-ethylmaleimide. CD analysis indicated that rBmG6PD is composed of 37% α-helices and 26% ß-sheets. The unfolding equilibrium of rBmG6PD with GdmCl/urea showed the triphasic unfolding pattern along with the highly stable intermediate obtained by GdmCl.

Intestinal smooth muscle cells locally enhance stem cell factor (SCF) production against gastrointestinal nematode infections.

Smooth muscle cells can produce stem cell factor (SCF) in the normal state for the preservation of mast cells, but it is still unknown whether smooth muscle cells can enhance SCF production in response to the pathological stimuli. The present study showed that smooth muscle cells in mast cell-increased regions around worm cysts of intestinal nematodes significantly enhanced SCF gene expression compared with mast cell non-increased regions in same sample. SCF gene expression in mast cell non-increased regions in nematode-infected mice showed almost the same level as in non-infected control groups. These results indicate that smooth muscle cells can locally enhance SCF gene expression, and may have a role in local immunological reactions as growth factor-producing cells.

Identification and expression of gene transcripts generated during an anti-parasitic response in Biomphalaria glabrata.

In order to gain further insights into the molecular basis of gastropod anti-parasite immune responses, we investigated transcripts of Biomphalaria glabrata regulated during hemocytic encapsulation. Using a snail strain that is resistant to the parasite Echinostoma caproni, we performed suppression subtractive hybridization (SSH) to construct cDNA libraries of transcripts more abundantly expressed in unexposed or parasite-exposed snails. After sequence analysis and quantitative PCR analysis of expression, we identified 10 candidates of particular interest. They belonged to various functional groups such as detoxification enzymes (GST, SOD), antimicrobial proteins (LBI/BPI), protease inhibitors (cystatins), calcium-binding proteins, or C-type lectins. In situ hybridization (ISH) analysis revealed that one overexpressed cystatin-like candidate is specifically expressed in hemocytes participating in parasite encapsulation or aggregating at the site of infection. Two other candidates (C-type lectin and a LBP/BPI) were expressed in the albumen gland, further supporting the role of this organ in immunity and/or host-parasite interaction.

Characterization of HC58cDNA, a putative cysteine protease from the parasite Haemonchus contortus.

Because of the complexity of the cathepsin B-like (CBL) family, an information on the biological and biochemical characteristics of individual CBL genes is lacking. In this study, we investigated the degradative effects of the recombinant HC58 protein isolated from Haemonchus contortus parasites on protein substrates over a broad pH range in vitro. This protein, which hydrolyzed the synthetic peptide substrates Z-FR-AMC and Z-RR-AMC, had characteristics of the cysteine protease class of proteins. In the acidic pH range, the isolated protein actively degraded hemoglobin (Hb), the heavy chain of goat immunoglobulin G, and azocasein. By contrast, it degraded fibrinogen in the alkaline pH range. These activities were strongly inhibited in the presence of the cysteine protease inhibitor E-64. While the protein digested Hb, it did not induce the agglutination of erythrocytes from its natural host. These results suggest that the HC58 protein may play a role in the nutrition of this parasite.

A membrane-associated metalloprotease of Taenia solium metacestode structurally related to the FACE-1/Ste24p protease family.

The CaaX proteases are intimately involved in the post-translational modification of prenylated proteins and play a critical role in the activation/stabilization of membrane-bound or secreted molecules constituting the CAAX protein family. In this study, we have isolated a full-length cDNA putatively encoding a type I CaaX protease of the Taenia solium metacestode (TsM), which an agent causative of human neurocysticercosis. The cDNA, designated TsSte24p, comprised 1,505 bp and coded for an open reading frame of 472 amino acids with predicted Mr 54.5 kDa. This monoexonic TsSte24p gene existed as a single copy within the TsM genome and constantly expressed in the parasite from metacestode to adult stages. The TsSte24p exhibited the typical CaaX protease topology, including seven transmembrane domains and a metalloprotease segment with a zinc-binding motif. It shared a significant degree of sequence identity with the type I CaaX proteases such as Saccharomyces cerevisiae Ste24p and Caenorhabditis elegans CeFACE-1. A comparative phylogenetic analysis demonstrated that this protein family is tightly conserved across taxa, from bacteria to mammals. The bacterially expressed recombinant TsSte24p showed proteolytic activity, with an optimal pH of 7.5. The enzyme activity was significantly inhibited by EDTA. Its activity was increased in the presence of low concentrations of the Zn2+(0.001-0.01 mM); but was reversibly down-regulated at high doses (over 0.1 mM). The native TsSte24p appeared to function as a homodimer, the subunits of which were linked to each other via covalent disulfide bond. The protein was localized in the bladder wall and scolex with differential patterns of distribution. Our results indicated that TsSte24p is a zinc-dependent metalloprotease, which belongs to the FACE-1/Ste24p protease family.

Mechanism and regulation of translation in C. elegans.

C. elegans represents a favorable system to study the extraordinarily complicated process of eukaryotic protein synthesis, which involves over 100 RNAs and over 200 polypeptides just for the core machinery. Initial research in protein synthesis relied on fractionated mammalian and plant systems, but in the mid-1970s, the powerful genetics of Saccharomyces cerevisiae began to yield new insights for translation in all eukaryotes. C. elegans has many features of higher eukaryotes that are not shared by yeast. This allows protein synthesis researchers to combine biochemistry, cell biology, developmental biology, genetics, and genomics to study regulation of gene expression at the translational level. Most components of the core translational machinery have been identified in C. elegans, including rRNAs, 5S RNA, tRNAs, ribosomal proteins, and aminoacyl tRNA synthetases. C. elegans has amino acid sequence homologs for 56 of the known initiation, elongation, and release factor polypeptides, but few of these have been isolated, functionally identified, or studied at the biochemical level. Similarly, C. elegans has homologs for 22 components of the major signal transduction pathways implicated in control of protein synthesis. The translational efficiency of individual mRNAs relies on cis-regulatory elements that include either a 7-methylguanosine- or 2,2,7-trimethylguanosine-containing cap, the 5'-terminal spliced leader, sequence elements in the 3'-untranslated regions, and the 3'-terminal poly(A) tract. Several key developmental pathways in C. elegans are predominantly governed by translational mechanisms. Some evidence has been presented that well described regulatory mechanisms in other organisms, including covalent modification of translation factors, sequestration of translation factors, and mRNA-specific changes in poly(A) length, also occur in C. elegans. The most interesting unexplored questions may involve changes in the translation of individual mRNAs during development, in response to physiological changes, or after genetic manipulations. Given the highly developed state of C. elegans genomics, it can be expected that future application of computational tools, including data visualization, will help detect new instances of translational control.

Modification at position 9 with 1-methyladenosine is crucial for structure and function of nematode mitochondrial tRNAs lacking the entire T-arm.

The mitochondria of the nematode Ascaris suum have tRNAs with unusual secondary structures that lack either the T-arm or D-arm found in most other organisms. Of the twenty-two tRNA species present in the mitochondria of A.suum, twenty lack the entire T-arm and two serine tRNAs lack the D-arm. To understand how such unusual tRNAs work in the nematode mitochondrial translation system, we analyzed post-transcriptional modifications of 11 mitochondrial tRNA species purified from A.suum, 10 of which lacked a T-arm and one of which lacked a D-arm. The most characteristic feature of nematode mitochondrial tRNAs lacking a T-arm was the presence of 1-methyladenosine at position 9 (m1A9). Synthesis of T-armless tRNAs with or without the modified nucleoside showed that T-armless tRNAs without the modification had much lower aminoacylation and EF-Tu-binding activities than native tRNAs. The addition of a single methyl group to A9 of these tRNAs was sufficient to restore nearly native levels of aminoacylation and EF-Tu-binding activity as well as tertiary structure, suggesting that m1A9 is a key residue for the activity of T-armless tRNAs. Thus, m1A9 is indispensable for the structure and function of T-armless tRNAs of nematode mitochondrial origin.

Expression of apoptosis-related factors in muscles infected with Trichinella spiralis.

We found that the expression of mitochondrial apoptosis related genes (Bcl-2 associated protein X, BAX; apoptotic protease activating factor 1, Apaf-1; Caspase 9 and serine/threonine protein kinase, PKB) is elevated in Trichinella spiralis-infected muscles during encapsulation. Micro-dissection of the capsule and subsequent reverse transcription polymerase chain reaction (RT-PCR) confirmed that the expressions of these genes are restricted to the nurse cell. Immunocytochemistry revealed that pro-apoptosis factor (BAX, Apaf-1 and Caspase 9) are predominantly expressed in the basophilic cytoplasm (infected muscle cell origin) and anti-apoptosis factor (PKB) in the eosinophilic cytoplasm (satellite cell origin) of the nurse cell. Electron microscopy revealed that the pre-existing mitochondria in the muscle cells became swollen and disappeared immediately after newborn larva invasion, but new mitochondria of smaller size appeared in the cytoplasm. Nuclear fragmentation and condensation were observed in basophilic cytoplasm which is known to die. Together, the results suggest that the infected muscle cells transform but die through the process of apoptosis which is triggered by factors from the newly formed mitochondria. The anti-apoptosis factor may help the eosinophilic cytoplasm with its survival to ensure nurse cell function.

Structural and evolutionary analysis of the transcribed sequence of Boudicca, a Schistosoma mansoni retrotransposon.

Boudicca is a gypsy-like, long terminal repeat (LTR) retrotransposon that has colonized the genome of the human blood fluke, Schistosoma mansoni. Previous studies have indicated that more than 1000 copies of Boudicca reside within the S. mansoni genome, although many of them may be degenerate and inactive. Messenger RNAs transcribed from genomic copies of Boudicca were investigated by reverse transcription PCR. Overlapping RT-PCR products corresponding to the gag and pol polyproteins of Boudicca, along with relevant sequences of genomic fragments of Boudicca, were assembled into contigs. Consensus sequences from these contigs were used to predict the sequence and structure of transpositionally active copies of the Boudicca retrotransposon. They verified that Boudicca has a kabuki-like Cys-His box motif at the active site of its gag protein, a classic DTG motif as the active site of the protease domain of the pol ORF2, and indicated a contiguous integrase domain at the C-terminus of pol with strong identity to integrase from the LTR retrotransposons CsRn1 and kabuki, as well as to the conserved integrase core domain, GenBank rve (). Models of the secondary structure of the Boudicca transcript suggested that the first AUG was occluded by a stem loop structure, which in turn suggested a method of regulation of expression, at the level of translation, of Boudicca proteins. In addition, phylogenetic analysis targeting discrete domains of Boudicca revealed a generalized radiation in sequences among the multiple copies of Boudicca resident in the schistosome genome.

Inhibition of bovine T lymphocyte responses by extracts of the stomach worm Ostertagia ostertagi.

Lowered immune responses during bovine ostertagiosis have been reported in both in vivo and in vitro assay systems. In the present study we have employed three different life cycle stages of the nematode Ostertagia ostertagi to determine if products of this economically important parasite inhibit in vitro proliferation of Con A-stimulated cells from uninfected animals. We have demonstrated an inhibitory effect upon the growth of Con A-stimulated lymphocytes after addition of fourth stage larval (L4) soluble extract (L4SE) to the cultures. In contrast, extracts from the third stage larvae (L3) had little or no inhibitory activity. The suppressive products were also shown to be secreted by the late L4. The suppressive activity is reversible if the L4 products are removed from culture. There is no immediate effect on proliferating cells and the L4SE must be in culture for 24-48 h before suppression is observable. The L4SE caused slight but not statistically significant decreases in the percentage of T cells and increases in B cell percentages in cultures when compared with cultures stimulated with Con A alone. No changes were seen in percentage of cells positive for markers for CD4, CD8, gammadelta T cells, or monocytes/macrophages as a consequence of the addition of L4SE. In contrast, there was a strong and significant reduction in the expression of the IL-2 receptors in cells cultured in the presence of the worm extract. There was no evidence of either necrosis or apoptosis resulting from the presence of L4 products in culture. The expression of messenger RNA for interleukin-2, -4, -13, tumor necrosis factor-alpha (TNF-alpha), and gamma-interferon (gamma-IFN) was decreased when L4SE was included in cultures of Con A-stimulated cells compared to cultures stimulated with Con A only. In contrast, messenger RNA expression of transforming growth factor-beta (TGF-beta) and interleukin-10 (IL-10) was increased in cells growing in the presence of L4 products. The potential role of these cytokines during ostertagiosis is discussed.

A cathepsin F of adult Clonorchis sinensis and its phylogenetic conservation in trematodes.

A novel 28 kDa cysteine protease (Cs28CF) secreted by the hepatobiliary trematode, Clonorchis sinensis was identified. The protease was purified from the excretory-secretory products (ESP) of the adult worm using DEAE-ion exchange and Arginine-Sepharose 4B chromatography. It showed a high activity between pH 6.5 and 7.5 in a dithiothreitol (DTT)-dependent manner. Inhibitors specific to cysteine proteases down-regulated the activity. Addition of Cs28CF to monkey cholangiocyte cultures resulted in approximately 95% cell death after 7 days. The full-length cDNA (1078 bp) encoded a single peptide of 328 amino acids (aa) with an N-terminal hydrophobic sequence, an ERFNAQ motif in the propeptide and a mature domain. Expression of mRNA transcripts of Cs28CF was observed in both the metacercaria and adult stages. Bacterially expressed recombinant protein exhibited a specific antibody reaction with clonorchiasis sera. Deduced aa exhibited 52-76% sequence identity with the cathepsin F analogues from other organisms. A novel E/DXGTA motif was recognized in the propeptide region. Phylogenetic analysis of 63 papain family members revealed that the trematode cysteine proteases formed 2 major clades of cathepsins F and L. The trematode cysteine proteases classified as cathepsin F shared higher homology among themselves than those classified as cathepsin L. Cathepsin F is phylogenetically conserved in the trematode parasites as well as in mammals.

Cloning of a pore-forming subunit of ATP-sensitive potassium channel from Clonorchis sinensis.

A complete cDNA sequence encoding a pore-forming subunit (Kir6.2) of ATP-sensitive potassium channel in the adult worm, Clonorchis sinensis, termed CsKir6.2, was isolated from an adult cDNA library. The cDNA contained a single open-reading frame of 333 amino acids, which has a structural motif (a GFG-motif) of the putative pore-forming loop of the Kir6.2. Peculiarly, the CsKir6.2 shows a lack-sequence structure, which deleted 57 amino acids were deleted from its N-terminus. The predicted amino acid sequence revealed a highly conserved sequence as other known other Kir6.2 subunits. The mRNA was weekly expressed in the adult worm.

Expression of cysteine proteinase of Clonorchis sinensis and its use in serodiagnosis of clonorchiasis.

A gene encoding cysteine proteinase from Clonorchis sinensis has been cloned and expressed in Escherichia coli. The cysteine proteinase cDNA fragment was amplified by reverse transcription-polymerase chain reaction using degenerate oligonucleotide primers derived from conserved active site of cysteine proteinases. The 5' and 3' regions of the gene were amplified using rapid amplification of cDNA ends. The cloned gene has an open reading frame of 696 bp and deduced amino acid sequence of 232. Sequence analysis and alignment showed significant homologies with the eukaryotic cysteine proteinases and conservation of the Cys, His, and Asp residues that form the catalytic triad. Analysis of the expressed protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the molecular weight of the protein was approximately 28.5 kDa. Proteolytic activity of the expressed protein was inhibited by cysteine proteinase inhibitors such as L-trans-epoxysuccinyl-leucylamide-(4-guanidino)-butane, iodoacetic acid, and leupeptin. The expressed protein showed biochemical properties similar to those of cysteine proteinases of other parasites. The expressed protein strongly reacted with the sera from patients with clonorchiasis but not with the sera from patients with paragonimiasis, fascioliasis, cysticercosis, and sparganosis, or with sera from normal human controls. These results suggest that the expressed protein may be valuable as a specific diagnostic material for the immunodiagnosis of clonorchiasis.

Purification and characterization of a 7-kDa protein from Clonorchis sinensis adult worms.

A 7-kDa protein was purified from extracts of adult Clonorchis sinensis by a combination of ammonium sulfate precipitation, anion exchange chromatography, cation exchange chromatography, gel-filtration chromatography, and reversed-phase FPLC. The 7-kDa protein exists in the excretory-secretory products of adult C. sinensis, but not in extracts of adult Paragonimus westermani. Also, the 7-kDa protein reacted with the sera of patients with clonorchiasis but not with paragonimiasis or normal human sera. To observe the localization of the 7-kDa protein in the tissue of adult C. sinensis, an immunogold labeling method was followed using anti-7-kDa antibody. The gold particles were observed in the basal layer below the tegumental syncytium, in the interstitial matrix of the parenchyma, and in the content of the uterus. The 7-kDa cDNA was obtained through reverse transcription-polymerase chain reaction using a primer designed from N-terminal sequence analysis. Rapid amplification of cDNA ends (5'-RACE) was used to obtain the complete protein coding sequence. The sequence encodes a 90-amino acid polypeptide. The deduced amino acid sequence of the 7-kDa protein revealed no homology with proteins of different organisms reported so far. These results suggest that the 7-kDa protein is a fluid antigen and may be valuable as a tool for the immunodiagnosis of clonorchiasis.

RNA hairpins in noncoding regions of human brain and Caenorhabditis elegans mRNA are edited by adenosine deaminases that act on RNA.

Adenosine deaminases that act on RNA (ADARs) constitute a family of RNA-editing enzymes that convert adenosine to inosine within double-stranded regions of RNA. We previously developed a method to identify inosine-containing RNAs and used it to identify five ADAR substrates in Caenorhabditis elegans. Here we use the same method to identify five additional C. elegans substrates, including three mRNAs that encode proteins known to affect neuronal functions. All 10 of the C. elegans substrates are edited in long stem-loop structures located in noncoding regions, and thus contrast with previously identified substrates of other organisms, in which ADARs target codons. To determine whether editing in noncoding regions was a conserved ADAR function, we applied our method to poly(A)+ RNA of human brain and identified 19 previously unknown ADAR substrates. The substrates were strikingly similar to those observed in C. elegans, since editing was confined to 3' untranslated regions, introns, and a noncoding RNA. Also similar to what was found in C. elegans, 15 of the 19 substrates were edited in repetitive elements. The identities of the newly identified ADAR substrates suggest that RNA editing may influence many biologically important processes, and that for many metazoa, A-to-I conversion in coding regions may be the exception rather than the rule.