Purification and characterization of a glycoprotein from Sipunculus nudus and its immune-enhancing activity to RAW 264.7 macrophages.

Sipunculus nudus, an edible marine invertebrate, has long been used as traditional Chinese medicine in folk remedies. In order to assess the immunoregulatory activity of glycoproteins in Sipunculus nudus and conduct a structure-activity relationship, a glycoprotein (SGP1) with molecular mass of 9.26 kDa was purified from Sipunculus nudus, and its chemical structure as well as immune-enhancing activity was investigated in this study. Structure analysis revealed that SGP1, a protein-dominate glycoprotein with O-glycosidic bonds, contained 92.8 % protein and 3.1 % saccharide. GC-MS result indicated that the saccharide moieties of SGP1 basically consisted of lyxose (Lyx), xylose (Xyl) as well as glucose (Glu) at a molar proportion of 0.87:4.16:1.36. The fourier transform infrared specoscopy (FT-IR) result proved that SGP1 have a typical characteristic of glycoprotein. Besides, circular dichroism (CD) result showed that SGP1 contained 4.1 % α-helix, 42.5 % ß-sheet, 21.4 % ß-turn, and 32.0 % random coil, indicating it's mainly a ß-sheet glycoprotein. The amino acid sequence of SGP1 shared a similarity to the Myohemerythrin (sp|Q5K473|HEMTM) with protein sequence coverage of 28.3 %. Moreover, the activity evaluation results showed that SGP1 exhibited significant immune-enhancing activity to the RAW 264.7 macrophages by promoting macrophages proliferation, enhancing phagocytic capacity, and simultaneously stimulating the secretions of nitric oxide (NO), interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) via NF-κB pathways. In this study, SGP1 as a novel glycoprotein had an obvious immune-enhancing activity to macrophages, and thus could be applied in the functional foods as a potential immunopotentiator for the hypoimmune population.

Nematode ascarosides attenuate mammalian type 2 inflammatory responses.

Mounting evidence suggests that nematode infection can protect against disorders of immune dysregulation. Administration of live parasites or their excretory/secretory (ES) products has shown therapeutic effects across a wide range of animal models for immune disorders, including asthma. Human clinical trials of live parasite ingestion for the treatment of immune disorders have produced promising results, yet concerns persist regarding the ingestion of pathogenic organisms and the immunogenicity of protein components. Despite extensive efforts to define the active components of ES products, no small molecules with immune regulatory activity have been identified from nematodes. Here we show that an evolutionarily conserved family of nematode pheromones called ascarosides strongly modulates the pulmonary immune response and reduces asthma severity in mice. Screening the inhibitory effects of ascarosides produced by animal-parasitic nematodes on the development of asthma in an ovalbumin (OVA) murine model, we found that administration of nanogram quantities of ascr#7 prevented the development of lung eosinophilia, goblet cell metaplasia, and airway hyperreactivity. Ascr#7 suppressed the production of IL-33 from lung epithelial cells and reduced the number of memory-type pathogenic Th2 cells and ILC2s in the lung, both key drivers of the pathology of asthma. Our findings suggest that the mammalian immune system recognizes ascarosides as an evolutionarily conserved molecular signature of parasitic nematodes. The identification of a nematode-produced small molecule underlying the well-documented immunomodulatory effects of ES products may enable the development of treatment strategies for allergic diseases.

The role of fish helminth parasites in monitoring metal pollution in aquatic ecosystems: a case study in the world's most productive platinum mining region.

Due to the increasing consumption of platinum (Pt), especially in automobile exhaust catalysts, environmental concentrations of Pt are of emerging concern worldwide. Limited information exists on environmental concentrations, particularly in Pt mining regions, while South Africa is the world's main supplier of Pt. Moreover, other metals are also released as by-products of Pt mining, which might also cause environmental concern. Certain fish parasite taxa have the ability to accumulate metals orders of magnitude higher than their hosts and can be used to reliably detect metals with naturally low abundance. Studies on Pt accumulation in parasite-host systems are limited. Therefore, the aims of the present study were (1) to determine the accumulation of a variety of metals (cadmium (Cd), chromium (Cr), copper (Cu), nickel (Ni), lead (Pb), platinum (Pt), and zinc (Zn)) in helminth fish parasites compared with their hosts from a reference site and an impoundment impacted by Pt mining activities; (2) to assess whether there is a difference between bioaccumulation of metals in infected and uninfected hosts, as well as between hosts with different infection intensities; and (3) to compare the biomarker responses (acetylcholine esterase activity (AChE), metallothionein content (MT), catalase activity (CAT), reduced glutathione content (GSH), malondialdehyde content (MDA), protein carbonyls induction (PC), superoxide dismutase activity (SOD), and cellular energy allocation (CEA)) between infected and uninfected hosts. The cestode Atractolytocestus huronensis accumulated significantly higher concentrations of Cr, Ni, and Pt than their host Cyprinus carpio, while the nematode Contracaecum sp. accumulated significantly higher concentrations of Pt and Zn than their host Clarias gariepinus. Infected fish showed lower metal concentrations compared to uninfected fish, while the parasites had no significant effects on their hosts' biomarker responses. The parasites demonstrated the bioavailability of metals derived from Pt mining activities and their ability to resist its toxic effects. Thus, these parasites are promising sensitive accumulation indicators for Cr, Ni, Pb, and Pt contaminations from Pt mining activities.

CCS52 and DEL1 function in root-knot nematode giant cell development in Xinjiang wild myrobalan plum (Prunus sogdiana Vassilcz).

Root-knot nematodes (RKNs) are highly invasive plant parasites that establish permanent feeding sites within the roots of the host plant. Successful establishment of the feeding site is essential for the survival of RKN. The formation and development of the feeding cell, also called giant cell, involve both cell division and endoreduplication. Here, we examined giant cell development and endoreduplication in Prunus sogdiana infected with the RKN. We found that feeding sites were established 3-5 days post inoculation (dpi) and matured at 21-28 dpi. The giant cells began to form 5 dpi and continued to increase in size from 7 to 21 dpi. The large numbers of dividing nuclei were observed in giant cells from 7 to 14 dpi. However, nuclear division was rarely observed after 28 days. RT-PCR and in situ hybridization analyses revealed that PsoCCS52A was abundantly expressed at 7-21 dpi and the PsoCCS52A signal observed in giant cell nucleus at 7-14 dpi. The PsoCCS52B is highly expressed at 14 dpi, and the hybridization signal was mainly in the cytoplasm of giant cells. The PsoDEL1 expression was lowest 7-21 dip, with negligible transcript detected in the giant cells. This indicates that the PsoCCS52A plays a role in the process of cell division, while the CCS52B plays a role in the development of giant cells. The PsoDEL1 plays a negative regulatory role in megakaryocyte nuclear replication. These data suggest that an increased expression of PsoCCS52A promotes nuclear division and produces a large number of polyploid nuclei, the area of giant cells and feeding sites increase, ultimately leading to the formation of galls in Prunus sogdiana.

Molecular Evolutionary Analysis of Nematode Zona Pellucida (ZP) Modules Reveals Disulfide-Bond Reshuffling and Standalone ZP-C Domains.

Zona pellucida (ZP) modules mediate extracellular protein-protein interactions and contribute to important biological processes including syngamy and cellular morphogenesis. Although some biomedically relevant ZP modules are well studied, little is known about the protein family's broad-scale diversity and evolution. The increasing availability of sequenced genomes from "nonmodel" systems provides a valuable opportunity to address this issue and to use comparative approaches to gain new insights into ZP module biology. Here, through phylogenetic and structural exploration of ZP module diversity across the nematode phylum, I report evidence that speaks to two important aspects of ZP module biology. First, I show that ZP-C domains-which in some modules act as regulators of ZP-N domain-mediated polymerization activity, and which have never before been found in isolation-can indeed be found as standalone domains. These standalone ZP-C domain proteins originated in independent (paralogous) lineages prior to the diversification of extant nematodes, after which they evolved under strong stabilizing selection, suggesting the presence of ZP-N domain-independent functionality. Second, I provide a much-needed phylogenetic perspective on disulfide bond variability, uncovering evidence for both convergent evolution and disulfide-bond reshuffling. This result has implications for our evolutionary understanding and classification of ZP module structural diversity and highlights the usefulness of phylogenetics and diverse sampling for protein structural biology. All told, these findings set the stage for broad-scale (cross-phyla) evolutionary analysis of ZP modules and position Caenorhabditis elegans and other nematodes as important experimental systems for exploring the evolution of ZP modules and their constituent domains.

Anti-inflammatory action of two novel peptides derived from peanut worms (Sipunculus nudus) in lipopolysaccharide-induced RAW264.7 macrophages.

Peanut worm (Sipunculus nudus Linn.) protein was hydrolyzed by three proteases, and NO scavenging activity of the protein hydrolysates was evaluated. The hydrolysate obtained using Alcalase® showed the highest NO scavenging activity. This hydrolysate was fractionated using 10-, 5-, and 3 kDa molecular weight cut-off membranes, and the lowest MW fraction (<3 kDa) exhibited the highest NO scavenging activity. The <3 kDa fraction was further purified by gel filtration and high-performance liquid chromatographies. The peptides in the HPLC fraction with the strongest anti-NO activity were identified by quadrupole-time-of-flight mass spectrometry as LSPLLAAH (821.48 Da) and TVNLAYY (843.42 Da). Both peptides and the corresponding pure synthetic peptides inhibited NO production by RAW 264.7 macrophages without cytotoxicity. Quantitative real-time RT-PCR analysis showed that peptides LSPLLAAH and TVNLAYY reduced expression of proinflammatory cytokine genes iNOS, IL-6, TNF-α, and COX-2 in RAW 264.7 macrophages, suggesting that these peptides are novel anti-inflammatory candidates.

Cysteine proteases secreted by the pinewood nematode, Bursaphelenchus xylophilus: In silico analysis.

The pinewood nematode, Bursaphelenchus xylophilus, is an important plant-parasitic nematode responsible for the development of the pine wilt disease and recognised as a major forest pest. Previous studies on the comparison of B. xylophilus and B. mucronatus secretomes obtained under maritime pine, Pinus pinaster, wood extract stimulus revealed that several cysteine proteases were increased in B. xylophilus secretome. In nematodes, proteases are known to play critical roles in parasitic processes like tissue penetration, digestion of host tissues for nutrition and evasion of host immune response. To gain further insight into the possible role of cysteine proteases on B. xylophilus pathogenicity, the molecular characterisation of four secreted cysteine peptidases was performed. BxCP3 and BxCP11 were identified as cathepsin L-like proteins and BxCP7 and BxCP8 as cathepsin B proteins. Only BxCP8 revealed high homology with another B. xylophilus cathepsin B referred on GenBank, all the others differ from the closer proteins deposited in this database. In silico three-dimensional structures of the four BxCP suggest that these proteins are pro-enzymes that become active when the pro-peptide is cleaved. BxCP7 and BxCP8 predicted structures revealed the presence of an occluding loop that occludes the active site cleft, typical of cathepsin B proteases.

Toxicity interactions between manganese (Mn) and lead (Pb) or cadmium (Cd) in a model organism the nematode C. elegans.

Manganese (Mn) is considered as an emerging metal contaminant in the environment. However, its potential interactions with companying toxic metals and the associated mixture effects are largely unknown. Here, we investigated the toxicity interactions between Mn and two commonly seen co-occurring toxic metals, Pb and Cd, in a model organism the nematode Caenorhabditis elegans. The acute lethal toxicity of mixtures of Mn+Pb and Mn+Cd were first assessed using a toxic unit model. Multiple toxicity endpoints including reproduction, lifespan, stress response, and neurotoxicity were then examined to evaluate the mixture effects at sublethal concentrations. Stress response was assessed using a daf-16::GFP transgenic strain that expresses GFP under the control of DAF-16 promotor. Neurotoxicity was assessed using a dat-1::GFP transgenic strain that expresses GFP in dopaminergic neurons. The mixture of Mn+Pb induced a more-than-additive (synergistic) lethal toxicity in the worm whereas the mixture of Mn+Cd induced a less-than-additive (antagonistic) toxicity. Mixture effects on sublethal toxicity showed more complex patterns and were dependent on the toxicity endpoints as well as the modes of toxic action of the metals. The mixture of Mn+Pb induced additive effects on both reproduction and lifespan, whereas the mixture of Mn+Cd induced additive effects on lifespan but not reproduction. Both mixtures seemed to induce additive effects on stress response and neurotoxicity, although a quantitative assessment was not possible due to the single concentrations used in mixture tests. Our findings demonstrate the complexity of metal interactions and the associated mixture effects. Assessment of metal mixture toxicity should take into consideration the unique property of individual metals, their potential toxicity mechanisms, and the toxicity endpoints examined.

Cysteine-Derived Pleurotin Congeners from the Nematode-Trapping Basidiomycete Hohenbuehelia grisea.

The discovery of a Hohenbuehelia grisea specimen during a field trip in Northern Thailand led to the isolation and identification of three novel sulfur-bearing derivatives of dihydropleurotinic acid (4). Thiopleurotinic acid A (1) was established by the interpretation of spectral data (HRESIMS, 2D-NMR) as a 2-hydroxy-3-mercaptopropanoic acid conjugate of dihydropleurotinic acid. Thiopleurotinic acid B (2) was shown to be the N-acetylcysteine conjugate of 4. A third compound (3) was established as a thiazole-containing derivative. Through feeding experiments with [U-13C3, 15N]-l-cysteine the formation of all three metabolites was shown to involve cysteine condensation with 4. The decreased cytotoxicity and antimicrobial activities of the new derivatives 1-3, compared to the parent compound 4, indicate a possible detoxification pathway of filamentous fungi.

Characterization of bactericidal efficiency, cell selectivity, and mechanism of short interspecific hybrid peptides.

Facing rising global antibiotics resistance, physical membrane-damaging antimicrobial peptides (AMPs) represent promising antimicrobial agents. Various strategies to design effective hybrid peptides offer many advantages in overcoming the adverse effects of natural AMPs. In this study, hybrid peptides from different species were investigated, and three hybrid antimicrobial peptides, LI, LN, and LC, were designed by combining the typical fragment of human cathelicidin-derived LL37 with either indolicidin, pig nematode cecropin P1 (CP-1) or rat neutrophil peptide-1 (NP-1). In an aqueous solution, all hybrid peptides had an unordered conformation. In simulated membrane conditions, the hybrid peptide LI displayed more ß-turn and ß-hairpin structures, whereas LN and LC folded into α-helix structures. The three interspecific hybrid peptides LI, LN, and LC exhibited different levels of antimicrobial activity against Gram-positive and Gram-negative bacteria. LI demonstrated the highest antimicrobial activity and cell selectivity. The results of the swimming motility indicated that LI repressed bacterial motility in a concentration-dependent method. Endotoxin binding assay demonstrated that hybrid peptide LI conserved the binding ability to LPS (polyanionic lipopolysaccharides) of its parental peptides. Fluorescence assays, flow cytometry, and SEM further revealed that hybrid peptide LI acted through different bacteriostatic mechanisms than LL37 and indolicidin and that LI killed bacterial cells via membrane damage. In summary, this study demonstrated that hybrid peptide LI produced by interspecific hybrid synthesis possessed strong cell selectivity and is a promising therapeutic candidate for drug-resistant bacteria infection.

Chrodrimanins O-S from the fungus Penicillium sp. SCS-KFD09 isolated from a marine worm, Sipunculusnudus.

Five new meroterpenoids, chrodrimanins O-S (1-5), as well as a known one (6), were isolated from the fermentation broth of Penicillium sp. SCS-KFD09 isolated from a marine worm, Sipunculusnudus, from Haikou Bay, China. The structures including the absolute configurations of the new compounds were unambiguously elucidated by spectroscopic data and ECD spectra analysis along with quantum ECD calculations. Among them, compound 1 represents the first example of an unusual trichlorinated meroterpenoid with an unique dichlorine functionality. Compounds 1 and 4-6 displayed inhibitory activity of protein tyrosine phosphatase 1B (PTP1B) with IC50 values of 71.6, 62.5, 63.1, and 39.6µM, respectively, and showed no apparent activity against three tumor cell lines (A549, HepG2, and Hela) and human umbilical vein endothelial cells (HUVEC) at 10µM.

Radioprotective effects of Sipunculus nudus L. polysaccharide combined with WR-2721, rhIL-11 and rhG-CSF on radiation-injured mice.

This study investigated the radioprotective effect of Sipunculus nudus L. polysaccharide (SNP) in combination with WR-2721, rhIL-11 and rhG-CSF on irradiated mice. A total of 70 Imprinting Control Region (ICR) mice were divided into seven groups: the control group, the model group and five administration groups. All groups, except the control group, were exposed to a 5 Gy (60)Co γ-ray beam. Blood parameters [including white blood cell (WBC), red blood cell (RBC) and platelet counts and hemoglobin level] were assessed three days before irradiation, and the on the 3rd, 7th and 14th days after irradiation. Spleen, thymus and testicular indices, DNA contents of bone marrow cells, bone marrow nucleated cells, sperm counts, superoxide dismutase (SOD), malondialdehyde (MDA), testosterone and estradiol levels in the serum were assessed on the 14th day after irradiation. The combined administration of SNP, WR-2721, rhIL-11 and rhG-CSF exerted synergistic recovery effects on peripheral blood WBC, RBC and platelet counts and hemoglobin levels in irradiated mice, and synergistic promotion effects on spleen, thymus, testicle, bone marrow nucleated cells and sperm counts in irradiated mice. The synergistic administration increased the serum SOD activities and serum testosterone content of irradiated mice, but synergy decreased the content of serum MDA and estradiol in irradiated mice. These results suggest that the combined administration of SNP, WR-2721, rhIL-11 and rhG-CSF should increase the efficacy of these drugs for acute radiation sickness, protect immunity, hematopoiesis and the reproductive organs of irradiated-damaged mice, and improve oxidation resistance in the body.

Soil nematode assemblages as bioindicators of radiation impact in the Chernobyl Exclusion Zone.

In radioecology, the need to understand the long-term ecological effects of radioactive contamination has been emphasised. This requires that the health of field populations is evaluated and linked to an accurate estimate of received radiological dose. The aim of the present study was to assess the effects of current radioactive contamination on nematode assemblages at sites affected by the fallout from the Chernobyl accident. First, we estimated the total dose rates (TDRs) absorbed by nematodes, from measured current soil activity concentrations, Dose Conversion Coefficients (DCCs, calculated using EDEN software) and soil-to-biota concentration ratios (from the ERICA tool database). The impact of current TDRs on nematode assemblages was then evaluated. Nematodes were collected in spring 2011 from 18 forest sites in the Chernobyl Exclusion Zone (CEZ) with external gamma dose rates, measured using radiophotoluminescent dosimeters, varying from 0.2 to 22 µGy h(-1). These values were one order of magnitude below the TDRs. A majority of bacterial-, plant-, and fungal-feeding nematodes and very few of the disturbance sensitive families were identified. No statistically significant association was observed between TDR values and nematode total abundance or the Shannon diversity index (H'). The Nematode Channel Ratio (which defines the relative abundance of bacterial- versus fungal-feeding nematodes) decreased significantly with increasing TDR, suggesting that radioactive contamination may influence nematode assemblages either directly or indirectly by modifying their food resources. A greater Maturity Index (MI), usually characterising better soil quality, was associated with higher pH and TDR values. These results suggest that in the CEZ, nematode assemblages from the forest sites were slightly impacted by chronic exposure at a predicted TDR of 200 µGy h(-1). This may be imputable to a dominant proportion of pollutant resistant nematodes in all sites. This might result from a selection at the expense of sensitive species after the accident.

Effects of Group 3 LEA protein model peptides on desiccation-induced protein aggregation.

Group 3 late embryogenesis abundant (G3LEA) proteins have amino acid sequences with characteristic 11-mer motifs and are known to reduce aggregation of proteins during dehydration. Previously, we clarified the structural and thermodynamic properties of the 11-mer repeating units in G3LEA proteins using synthetic peptides composed of two or four tandem repeats originating from an insect (Polypedilum vanderplanki), nematodes and plants. The purpose of the present study is to test the utility of such 22-mer peptides as protective reagents for aggregation-prone proteins. For lysozyme, desiccation-induced aggregation was abrogated by low molar ratios of a 22-mer peptide, PvLEA-22, derived from a P. vanderplanki G3LEA protein sequence. However, an unexpected behavior was noted for the milk protein, α-casein. On drying, the resultant aggregation was significantly suppressed in the presence of PvLEA-22 with its molar ratios>25 relative to α-casein. However, when the molar ratio was <10, aggregation occurred on addition of PvLEA-22 to aqueous solutions of α-casein. Other peptides derived from nematode, plant and randomized G3LEA protein sequences gave similar results. Such an anomalous solubility change in α-casein was shown to be due to a pH shift to ca. 4, a value nearly equal to the isoelectric point (pI) of α-casein, when any of the 22-mer peptides was mixed. These results demonstrate that synthetic peptides derived from G3LEA protein sequences can reduce protein aggregation caused both by desiccation and, at high molar ratios, also by pH effects, and therefore have potential as stabilization reagents.

The parasitic nematodes Hysterothylacium sp. type MB larvae as bioindicators of lead and cadmium: a comparative study of parasite and host tissues.

Cadmium and lead concentrations were compared in tissues of cutlassfish, Trichiurus lepturus L., its intestinal nematode Hysterothylacium sp. type MB larvae, and in water from the same location in the Sea of Oman. Metal accumulation in hosts, parasites and sea water was measured by ICP-OES. Hysterothylacium larvae from the intestinal lumen and visceral cavity showed much higher metal concentrations than in host tissues or sea water. Statistical analyses revealed no significant differences in metal accumulation between infected and uninfected hosts. Cadmium concentration in the host muscle was lower than in intestine, liver and gonad tissues. The mean concentrations of lead and cadmium in nematodes were 289·03 and 81·5 times higher than in host intestine, 188·4 and 225 times higher than in host muscle, 108·6 and 65·3 times higher than in host gonads, 70·5 and 19·5 times higher than in host liver and 3351 and 148 times higher than in sea water. The results show the value of this and possibly related nematodes as bioindicators of heavy metals and their potential use in environmental studies.

Anti-inflammatory and anti-nociceptive activities of Sipunculus nudus L. extract.

ETHNOPHARMACOLOGICAL RELEVANCE: Sipunculus nudus has long been employed as traditional Chinese medicine in folk remedies for the treatment of carbuncles, tuberculosis and nocturia, regulating the functions of stomach and spleen, as well as for the restoration to health in debilities caused by various pathogens and aging. Decoction of Sipunculus nudus has traditionally been used to remedy sternalgia in folk medicine. AIM OF THE STUDY: This study aimed to assess the anti-inflammatory and anti-nociceptive activity of the water extract from Sipunculus nudus. MATERIALS AND METHODS: The water extract from the body wall of Sipunculus nudus was obtained with the yield of 14.1%. The anti-inflammatory effect in six animal models and anti-nociceptive effect in two animal models of the water extract were evaluated by oral for the study. RESULTS: Pretreatment with the extract (at the dose of 50, 100 and 200 mg/kg) produced significant dose-dependent anti-inflammatory and anti-nociceptive effects. At 200 mg/kg dose, the inhibition ratio of the extract on carrageenan-induced rat hind paw oedema, dextran-induced rat paw oedema, cotton pellet granuloma in rats, carrageenan-induced peritonitis and acetic acid-induced vascular permeability were 59.2%, 51.0%, 53.1%, 42.5%, 50.8%, better than that of indomethacin (5 mg/kg) 49.5%, 50%, 44.4%, 37.6%, 46.8%, respectively. The inhibition ratio of the extract (200 mg/kg dose) on xylene-induced mouse ear oedema was 61.5%, lower than 63.7% for indomethacin (5 mg/kg). At 200mg/kg, the extract decreased number of writhing 52.3% in acetic acid-induced writhing model and increased the response latency 25.24% in hot plate test. CONCLUSIONS: The water extract from the body wall of Sipunculus nudus possesses excellent anti-inflammatory activity as well as peripheral and central analgesic properties.

The polyprotein allergens of nematodes (NPAs) - structure at last, but still mysterious.

We are engaged in structural and functional studies of several types of lipid binding protein that are only found in nematodes. Amongst these are the nematode polyprotein allergens (NPAs) and we now report the solution structure of ABA-1A (As-NPA-A1), the most repeated unit within the NPA array of Ascaris suum, which is almost identical in amino acid sequence to that of Ascaris lumbricoides. The protein forms a slightly flattened, compact, globular fold consisting of a long central helix that participates in two flanking helical bundles. Two pockets lined with apolar amino acid sidechains are apparent, one in the carboxy-terminal region of the protein, and another smaller one in the amino-terminal region. The former appears to be the main site of fatty acid binding, and the latter may have different, though possibly overlapping, ligand binding propensities. The structure of the binding sites indicates that lipid ligands are anchored within them with their hydrophobic tails oriented towards the core of the protein and their polar headgroups bound to charged sidechains at the mouth of the pockets. The three-dimensional architectures of the amino- and carboxy-terminal halves of ABA-1A are closely similar, thereby strengthening the long-suspected idea that the repeated units of NPAs themselves originate from an ancient duplication event.

Conserved eukaryotic fusogens can fuse viral envelopes to cells.

Caenorhabditis elegans proteins AFF-1 and EFF-1 [C. elegans fusion family (CeFF) proteins] are essential for developmental cell-to-cell fusion and can merge insect cells. To study the structure and function of AFF-1, we constructed vesicular stomatitis virus (VSV) displaying AFF-1 on the viral envelope, substituting the native fusogen VSV glycoprotein. Electron microscopy and tomography revealed that AFF-1 formed distinct supercomplexes resembling pentameric and hexameric "flowers" on pseudoviruses. Viruses carrying AFF-1 infected mammalian cells only when CeFFs were on the target cell surface. Furthermore, we identified fusion family (FF) proteins within and beyond nematodes, and divergent members from the human parasitic nematode Trichinella spiralis and the chordate Branchiostoma floridae could also fuse mammalian cells. Thus, FF proteins are part of an ancient family of cellular fusogens that can promote fusion when expressed on a viral particle.

Evolution of GHF5 endoglucanase gene structure in plant-parasitic nematodes: no evidence for an early domain shuffling event.

BACKGROUND: Endo-1,4-beta-glucanases or cellulases from the glycosyl hydrolase family 5 (GHF5) have been found in numerous bacteria and fungi, and recently also in higher eukaryotes, particularly in plant-parasitic nematodes (PPN). The origin of these genes has been attributed to horizontal gene transfer from bacteria, although there still is a lot of uncertainty about the origin and structure of the ancestral GHF5 PPN endoglucanase. It is not clear whether this ancestral endoglucanase consisted of the whole gene cassette, containing a catalytic domain and a carbohydrate-binding module (CBM, type 2 in PPN and bacteria) or only of the catalytic domain while the CBM2 was retrieved by domain shuffling later in evolution. Previous studies on the evolution of these genes have focused primarily on data of sedentary nematodes, while in this study, extra data from migratory nematodes were included. RESULTS: Two new endoglucanases from the migratory nematodes Pratylenchus coffeae and Ditylenchus africanus were included in this study. The latter one is the first gene isolated from a PPN of a different superfamily (Sphaerularioidea); all previously known nematode endoglucanases belong to the superfamily Tylenchoidea (order Rhabditida). Phylogenetic analyses were conducted with the PPN GHF5 endoglucanases and homologous endoglucanases from bacterial and other eukaryotic lineages such as beetles, fungi and plants. No statistical incongruence between the phylogenetic trees deduced from the catalytic domain and the CBM2 was found, which could suggest that both domains have evolved together. Furthermore, based on gene structure data, we inferred a model for the evolution of the GHF5 endoglucanase gene structure in plant-parasitic nematodes. Our data confirm a close relationship between Pratylenchus spp. and the root knot nematodes, while some Radopholus similis endoglucanases are more similar to cyst nematode genes. CONCLUSION: We conclude that the ancestral PPN GHF5 endoglucanase gene most probably consisted of the whole gene cassette, i.e. the GHF5 catalytic domain and the CBM2, rather than that it evolved by domain shuffling. Our evolutionary model for the gene structure in PPN GHF5 endoglucanases implies the occurrence of an early duplication event, and more recent gene duplications at genus or species level.

RAWUL: a new ubiquitin-like domain in PRC1 ring finger proteins that unveils putative plant and worm PRC1 orthologs.

BACKGROUND: Polycomb group (PcG) proteins are a set of chromatin-modifying proteins that play a key role in epigenetic gene regulation. The PcG proteins form large multiprotein complexes with different activities. The two best-characterized PcG complexes are the PcG repressive complex 1 (PRC1) and 2 (PRC2) that respectively possess histone 2A lysine 119 E3 ubiquitin ligase and histone 3 lysine 27 methyltransferase activities. While PRC2-like complexes are conserved throughout the eukaryotic kingdoms, PRC1-like complexes have only been described in Drosophila and vertebrates. Since both complexes are required for the gene silencing mechanism in Drosophila and vertebrates, how PRC1 function is realized in organisms that apparently lack PRC1 such as plants, is so far unknown. In vertebrates, PRC1 includes three proteins, Ring1B, Ring1A, and Bmi-1 that form an E3 ubiquitin ligase complex. These PRC1 proteins have an N-terminally located Ring finger domain associated to a poorly characterized conserved C-terminal region. RESULTS: We obtained statistically significant evidences of sequence similarity between the C-terminal region of the PRC1 Ring finger proteins and the ubiquitin (Ubq)-like family proteins, thus defining a new Ubq-like domain, the RAWUL domain. In addition, our analysis revealed the existence of plant and worm proteins that display the conserved combination of a Ring finger domain at the N-terminus and a RAWUL domain at the C-terminus. CONCLUSION: Analysis of the conserved domain architecture among PRC1 Ring finger proteins revealed the existence of long sought PRC1 protein orthologs in these organisms, suggesting the functional conservation of PRC1 throughout higher eukaryotes.