Glycomics analysis of Schistosoma mansoni egg and cercarial secretions

The parasitic helminth Schistosoma mansoni is a major public health concern in many developing countries. Glycoconjugates, and in particular the carbohydrate component of these products, represent the main immunogenic challenge to the host and could therefore represent one of the crucial determinants for successful parasite establishment. Here we report a comparative glycomics analysis of the N- and O-glycans derived from glycoproteins present in S. mansoni egg (egg-secreted protein) and cercarial (0-3-h released protein) secretions by a combination of mass spectrometric techniques. Our results show that S. mansoni secrete glycoproteins with glycosylation patterns that are complex and stage-specific. Cercarial stage secretions were dominated by N-glycans that were core-xylosylated, whereas N-glycans from egg secretions were predominantly core-difucosylated. O-Glycan core structures from cercarial secretions primarily consisted of the core sequence Galbeta1-->3(Galbeta1-->6)GalNAc, whereas egg-secreted O-glycans carried the mucin-type core 1 (Galbeta1-->3GalNAc) and 2 (Galbeta1-->3(GlcNAcbeta1-->6)GalNAc) structures. Additionally we identified a novel O-glycan core in both secretions in which a Gal residue is linked to the protein. Terminal structures of N- and O-glycans contained high levels of fucose and include stage-specific structures. These glycan structures identified in S. mansoni secretions are potentially antigenic motifs and ligands for carbohydrate-binding proteins of the host immune system.     

Proteomic analyses of amniotic fluid: potential applications in health and diseases

Amniotic fluid (AF) is a potential source of biomarkers for many disorders which may occur during pregnancy. The purpose of this study was to evaluate the place of two-dimensional gel electrophoresis (2-DE) technologies to compare AF in both normal and pathological situations. Two-dimensional fluorescence difference gel electrophoresis (2D-DIGE; Ettan DIGE) as well as two-dimensional gel electrophoresis and silver staining followed by image analysis were used. Differentially expressed proteins were identified by mass spectrometry. This approach was used to study electrophoregrams of normal AF obtained at 17 weeks of gestation and at term, as well as AF from fetuses presenting with congenital diaphragmatic hernia. Finally, the potential of two-dimensional electrophoresis was assessed by studying the protein profile of plasma containing AF proteins in a model of premature rupture of the membranes (PROM). Our results clearly show that two-dimensional electrophoresis technologies still have place for analyzing biological fluids such as AF.     

Inhibition of gut- and lung-derived serotonin attenuates pulmonary hypertension in mice

Decreasing the bioavailability of serotonin (5-HT) by inhibiting its biosynthesis may represent a useful adjunctive treatment of pulmonary hypertension (PH). We assessed this hypothesis using LP533401, which inhibits the rate-limiting enzyme tryptophan hydroxylase 1 (Tph1) expressed in the gut and lung, without inhibiting Tph2 expressed in neurons. Mice treated repeatedly with LP533401 (30-250 mg/kg per day) exhibited marked 5-HT content reductions in the gut, lungs, and blood, but not in the brain. After a single LP533401 dose (250 mg/kg), lung and gut 5-HT contents decreased by 50%, whereas blood 5-HT levels remained unchanged, suggesting gut and lung 5-HT synthesis. Treatment with the 5-HT transporter (5-HTT) inhibitor citalopram decreased 5-HT contents in the blood and lungs but not in the gut. In transgenic SM22-5-HTT+ mice, which overexpress 5-HTT in smooth muscle cells and spontaneously develop PH, 250 mg/kg per day LP533401 or 10 mg/kg per day citalopram for 21 days markedly reduced lung and blood 5-HT levels, right ventricular (RV) systolic pressure, RV hypertrophy, distal pulmonary artery muscularization, and vascular Ki67-positive cells (P < 0.001). Combined treatment with both drugs was more effective in improving PH-related hemodynamic parameters than either drug alone. LP533401 or citalopram treatment partially prevented PH development in wild-type mice exposed to chronic hypoxia. Lung and blood 5-HT levels were lower in hypoxic than in normoxic mice and decreased further after LP533401 or citalopram treatment. These results provide proof of concept that inhibiting Tph1 may represent a new therapeutic strategy for human PH.     

Functional dissection of the Tol2 transposable element identified the minimal cis-sequence and a highly repetitive sequence in the subterminal region essential for transposition

The Tol2 element is a naturally occurring active transposable element found in vertebrate genomes. The Tol2 transposon system has been shown to be active from fish to mammals and considered to be a useful gene transfer vector in vertebrates. However, cis-sequences essential for transposition have not been characterized. Here we report the characterization of the minimal cis-sequence of the Tol2 element. We constructed Tol2 vectors containing various lengths of DNA from both the left (5') and the right (3') ends and tested their transpositional activities both by the transient excision assay using zebrafish embryos and by analyzing chromosomal transposition in the zebrafish germ lineage. We demonstrated that Tol2 vectors with 200 bp from the left end and 150 bp from the right end were capable of transposition without reducing the transpositional efficiency and found that these sequences, including the terminal inverted repeats (TIRs) and the subterminal regions, are sufficient and required for transposition. The left and right ends were not interchangeable. The Tol2 vector carrying an insert of >11 kb could transpose, but a certain length of spacer, <276 but >18 bp, between the left and right ends was necessary for excision. Furthermore, we found that a 5-bp sequence, 5'-(A/G)AGTA-3', is repeated 33 times in the essential subterminal region. Mutations in the repeat sequence at 13 different sites in the subterminal region, as well as mutations in TIRs, severely reduced the excision activity, indicating that they play important roles in transposition. The identification of the minimal cis-sequence of the Tol2 element and the construction of mini-Tol2 vectors will facilitate development of useful transposon tools in vertebrates. Also, our study established a basis for further biochemical and molecular biological studies for understanding roles of the repetitive sequence in the subterminal region in transposition.     

The Glycine-Rich Motif of Pyrococcus abyssi DNA Polymerase D Is Critical for Protein Stability

A glycine-rich motif described as being involved in human polymerase δ proliferating cell nuclear antigen (PCNA) binding has also been identified in all euryarchaeal DNA polymerase D (Pol D) family members. We redefined the motif as the (G)-PYF box. In the present study, Pol D (G)-PYF box motif mutants from Pyrococcus abyssi were generated to investigate its role in functional interactions with the cognate PCNA. We demonstrated that this motif is not essential for interactions between PabPol D (P. abyssi Pol D) and PCNA, using surface plasmon resonance and primer extension studies. Interestingly, the (G)-PYF box is located in a hydrophobic region close to the active site. The (G)-PYF box mutants exhibited altered DNA binding properties. In addition, the thermal stability of all mutants was reduced compared to that of wild type, and this effect could be attributed to increased exposure of the hydrophobic region. These studies suggest that the (G)-PYF box motif mediates intersubunit interactions and that it may be crucial for the thermostability of PabPol D.     

Corticosteroid effects on ventilator-induced diaphragm dysfunction in anesthetized rats depend on the dose administered

Background

High dose of corticosteroids has been previously shown to protect against controlled mechanical ventilation (CMV)-induced diaphragmatic dysfunction while inhibiting calpain activation. Because literature suggests that the calpain inhibiting effect of corticosteroid depends on the dose administered, we determined whether lower doses of corticosteroids would also provide protection of the diaphragm during CMV. This may be important for patients undergoing mechanical ventilation and receiving corticosteroids.

Methods

Rats were assigned to controls or to 24 hours of CMV while being treated at the start of mechanical ventilation with a single intramuscular administration of either saline, or 5 mg/kg (low MP) or 30 mg/kg (high MP) of methylprednisolone.

Results

Diaphragmatic force was decreased after CMV and this was exacerbated in the low MP group while high MP rescued this diaphragmatic dysfunction. Atrophy was more severe in the low MP group than after CMV while no atrophy was observed in the high MP group. A significant and similar increase in calpain activity was observed in both the low MP and CMV groups whereas the high dose prevented calpain activation. Expression of calpastatin, the endogenous inhibitor of calpain, was decreased in the CMV and low MP groups but its level was preserved to controls in the high MP group. Caspase-3 activity increased in all CMV groups but to a lesser extent in the low and high MP groups. The 20S proteasome activity was increased in CMV only.

Conclusions

Administration of 30 mg/kg methylprednisolone during CMV protected against CMV-induced diaphragm dysfunction while 5 mg/kg was more deleterious. The protective effect is due mainly to an inhibition of the calpain system through preservation of calpastatin levels and to a lesser extent to a caspase-3 inhibition.     

Larval connectivity in an effective network of marine protected areas

Acceptance of marine protected areas (MPAs) as fishery and conservation tools has been hampered by lack of direct evidence that MPAs successfully seed unprotected areas with larvae of targeted species. For the first time, we present direct evidence of large-scale population connectivity within an existing and effective network of MPAs. A new parentage analysis identified four parent-offspring pairs from a large, exploited population of the coral-reef fish Zebrasoma flavescens in Hawai'i, revealing larval dispersal distances ranging from 15 to 184 km. In two cases, successful dispersal was from an MPA to unprotected sites. Given high adult abundances, the documentation of any parent-offspring pairs demonstrates that ecologically-relevant larval connectivity between reefs is substantial. All offspring settled at sites to the north of where they were spawned. Satellite altimetry and oceanographic models from relevant time periods indicated a cyclonic eddy that created prevailing northward currents between sites where parents and offspring were found. These findings empirically demonstrate the effectiveness of MPAs as useful conservation and management tools and further highlight the importance of coupling oceanographic, genetic, and ecological data to predict, validate and quantify larval connectivity among marine populations.     

Clinical proteomics: study of a cryogel

Cryoproteins are proteins precipitating at low temperature. Usually, the precipitate contains immunoglobulins (Igs), and are therefore called cryoglobulins. Very rarely, Igs do not precipitate, but, upon cooling, form a gel. Here, we report a case of cryogel observed in a patient presenting with Waldenström's disease. Using proteomic tools, a monoclonal IgM was identified as being the cause of the gel formation. Furthermore, addition of H₂O before incubation at 4°C demonstrated that the monoclonal IgM was precipitable as a type I cryoglobulin (hypocryoglobulin).