Thrombocytopenia is a common symptom and one of the warning signs of dengue virus (DENV) infection. Platelet depletion is critical as it may lead to other severe dengue symptoms. Understanding the molecular events of this condition during dengue infection is challenging because of the multifaceted factors involved in DENV infection and the dynamics of the disease progression. Platelet levels depend on the balance between platelet production and platelet consumption or clearance. Megakaryopoiesis and thrombopoiesis, two interdependent processes in platelet production, are hampered during dengue infection. Conversely, platelet elimination via platelet activation, apoptosis and clearance processes are elevated. Together, these anomalies contribute to thrombocytopenia in dengue patients. Targeting the molecular events of dengue-mediated thrombocytopenia shows great potential but still requires further investigation. Nonetheless, the application of new knowledge in this field, such as immature platelet fraction analysis, may facilitate physicians in monitoring the progression of the disease.

The present study was designed to evaluate the effect of the combination of oviduct fluid flush (OFF) and oviduct epithelial cells (OEC) in modulating the incidence of polyspermy in pigs. Therefore, for in vitro fertilization (IVF), oocyte and sperm were co-cultured in Tris-buffered medium (TBM) either supplemented with 10% OFF (OFFD group), or in the presence of a bovine OEC monolayer (OEC group), or the oocytes were exposed to OFF for 30 min before IVF (OFFB group), or in the presence of an OEC monolayer (OFFB + OEC group). Regardless of sperm concentration used (0.5, 1.5, and 4.5 × 105 cells/ml), supplementation of IVF medium with 10% OFF led to an increased (P < 0.05) monospermy rate, without alteration (P > 0.05) of the penetration rate in comparison with the control and OEC groups. When the IVF medium was supplemented with heparin, an overall increase (P < 0.05) of the final output of the IVF system in terms of zygotes with two pronuclei (2PN) was observed in the OFFD group, compared with the control and OEC groups, at a sperm concentration of 4.5 × 105 cells/ml. At this concentration, OFFB improved the monospermy rate but decreased the penetration rate, resulting in low efficiency of monospermic zygotes production. Despite this, no major effect was observed in the developmental competence of the presumed zygotes up to the blastocyst stage. The combination of OFFB with OEC improved the penetration rate, while maintaining the high monospermic rate induced by OFFB. In conclusion, the combination of treatment of oocytes by diluted OFF 30 min before IVF, followed by IVF in the presence of OEC, improved monospermic zygote production without reducing the penetration rate, when the IVF medium was supplemented with heparin.

In the present study, we analyse the effect of glycosylation in Echinostoma caproni (Trematoda: Echinostomatidae) antigens in antibody responses against the parasite in experimentally infected mice. It has been previously demonstrated that the mouse is a host of high compatibility with E. caproni and develops elevated responses of IgG, IgG1, IgG3 and IgM as a consequence of the infection, though the role of glycans in these responses remains unknown. To this purpose, the responses generated in mice against non-treated excretory/secretory antigens of E. caproni were compared with those observed after N-deglycosylation, O-deglycosylation and double deglycosylation of the antigens by indirect ELISA and western blot. Our results suggest that E. caproni-expressed glycans play a major role in the modulation of the immune responses. The results obtained indicate that IgG subclass responses generated in mice against E. caproni are essentially due to glycoproteins and may affect the Th1/Th2 biasing. The reactivity significantly decreased after any of the deglycosylation treatments and the N-glycans appears to be of greater importance than O-glycans. Interestingly, the IgM response increased after N-deglycosylation suggesting that carbohydrates may mask peptide antigens.

Sirtuin proteins form a family of NAD+-dependent protein deacetylases that are considered potential drug targets against parasites. Here, we present the first characterization of a sirtuin orthologue from Leishmania amazonensis, an aetiological agent of American tegumentary leishmaniasis that has been the subject of many studies focused in the development of therapeutic approaches. The protein has high sequence identity with other Kinetoplastid Silent information regulator 2 Related Protein 1 (Sir2RP1) and was named LaSir2RP1. The gene exists as a single copy, encoding a monomeric protein (LaSir2RP1) of approximately 41 kDa that has NAD+-dependent deacetylase activity. LaSir2RP1 was immunodetected in total protein extracts, in cytoplasmic granules, and in the secreted material of both promastigotes and lesion-derived amastigotes. Analysis of both lectin‑affinity purified promastigote and amastigote extracts revealed the presence of a major enriched protein of approximately 66 kDa that was recognized by an anti-LaSir2RP1 serum, suggesting that a parasite sirtuin could be glycosylated in vivo.

Introduction. Leishmania donovani, the causative agent of visceral leishmaniasis in the Indian subcontinent, has been reported to be genetically homogeneous. In order to support ongoing initiatives to eliminate the disease, highly discriminative tools are required for documenting the parasite population and dynamics. Methods. Thirty-four clinical isolates of L. donovani from Nepal were analysed on the basis of size and restriction endonuclease polymorphisms of PCR amplicons from kinetoplast minicircle DNA, 5 nuclear microsatellites, and nuclear loci encoding glycoprotein 63, cysteine proteinase B, and hydrophilic acylated surface protein B. We present and validate a procedure allowing standardized analysis of kDNA fingerprint patterns. Results. Our results show that parasites are best discriminated on the basis of kinetoplast minicircle DNA (14 genotypes) and 1 microsatellite defining 7 genotypes, while the remaining markers discriminated 2 groups or were monomorphic. Combination of all nuclear markers revealed 8 genotypes, while extension with kDNA data yielded 18 genotypes. Conclusion. We present tools that allow discrimination of closely related L. donovani strains circulating in the Terai region of Nepal. These can be used to study the micro-epidemiology of parasite populations, determine the geographical origin of infections, distinguish relapses from re-infection, and monitor the spread of particular variants.

Employing papain as the enzyme and agarose bound Ricinus communis agglutinin as the affinity gel, a glycoprotein has been isolated and purified from the surface of Ascaridia galli. The glycoprotein shows an apparent molecular weight of 68 kilo daltons and contains fucose, galactose, rhamnose and glucosamine as sugar moieties. Only 2% of its entire molecule has been found to possess α-helical configuration.

A monoclonal antibody (OW-IGO) raised against purified excysted oocysts of Cryptosporidium parvum reacted in an immunofluorescence assay with the oocyst wall. The corresponding antigen was localized by immunoelectron microscopy in fibrillous material present in the parasitophorous vacuole of developing macrogametes and in the wall of both single and double layered sporulating oocysts. Gold particles were also detected over electron-lucent vesicles of the macrogametes by immunoelectron microscopy. On Western blotting of C. parvum oocyst extracts, major bands at 250 and 40 kDa and several minor components were recognized by Mab OW-IGO. Almost complete abolition of Western blot reactivity occurred after periodate oxidation of oocyst antigen, suggesting that monoclonal antibody OW-IGO reacts with a carbohydrate epitope. Taken together, our results suggest that a fibrillous glycoproteinic material is released in the parasitophorous vacuole from electron-lucent vesicles during gametogenesis, and later condensed in the oocyst wall.

Lagos bat virus (LBV) constitutes genotype (gt) 2 in the Lyssavirus genus. In contrast to the gt1 lyssavirus, rabies virus (RABV), LBV was reported to have markedly reduced levels of peripheral pathogenicity. However, this opinion was based on a study of one isolate of LBV only and the reduction in pathogenicity was essentially attributed to the amino-acid substitution at position 333 of glycoprotein ectodomain. In the present study we have demonstrated that peripheral pathogenicity of representatives of LBV in a murine model is as high as that of RABV. Comparison of amino-acid substitutions among the viral glycoproteins, demonstrated significant differences within two antigenic sites between different phylogenetic lineages of LBV. Such molecular variability potentially contributes to differences in peripheral pathogenicity of lyssaviruses.

In the starfish, spermatozoa undergo the acrosome reaction upon encountering the jelly coat of eggs. A highly sulphated glycoprotein in the jelly coat is called acrosome-reaction-inducing substance (ARIS) because it is the key signal molecule to trigger the acrosome reaction. The activity of ARIS is mainly attributed to its sulphate and saccharide residues. The extremely large molecular size and speciesspecific action of ARIS suggest the presence of a specific ARIS receptor on the sperm surface, but no experimental evidence for the receptor has been presented. We therefore measured specific binding of ARIS and its pronase digest (P-ARIS), which retains the full activity of ARIS, to homologous spermatozoa by using fluorescien-isothiocyanate-labelled ARIS and125 I-labelled P-ARIS, respectively. The spermatozoa had the ability to bind ARIS, as well as P-ARIS, specifically. The binding was species-specific, and mostly localised to the head region of spermatozoa. Scatchard plot analysis indicated the presence of one class of ARIS receptor on the surface of acrosome-intact speramatozoa. Furthermore, the specific binding of P-ARIS to the anterior region of sperm heads was microscopically confirmed by using P-ARIS conjugated to polystyrene latex beads with intense fluorescence. It is concluded that starfish spermatozoa have a specific receptor for ARIS on the surface of the anterior region of heads.

TEX101, a glycoprotein we recently identified, is primarily characterized as a unique germ-cell-specific marker protein that shows sexually dimorphic expression during mouse gonad development. Based on data obtained from molecular biological as well as immuno-morphological studies, we believe this molecule may play a role in the process underlying germ cell formation. However, many points remain unclear as the molecular characteristics and its physiological functions are far from being completely understood. To clarify the molecular basis of TEX101, we herein report a further biochemical characterization of the molecule using testicular Triton X-100 extracts from mice. Deglycosylation studies using endoglycohydrolases that delete N-linked oligosaccharides (OS) from the molecule show that TEX101 is highly (approximately 47%) N-glycosylated. All potential N-glycosylation sites within TEX101 are glycosylated and most of these sites are occupied by endoglycosidase F2-sensitive biantennary complex type OS units. In addition, an extremely low population among TEX101 possesses only endoglycosidase H-sensitive hybrid type OS units. In studies using phosphatidylinositol-specific phospholipase C against native testicular cells or TEX101 transfectant, the enzyme treatment caused major reduction of the TEX101 expression on the cell, suggesting that TEX101, at least in part, is expressed as a glycosylphosphatidylinositol-anchored protein. Taken together, these findings will help elucidate the molecular nature of TEX101, a marker molecule that appeared on germ cells during gametogenesis.

It has been reported that various structural and functional changes occur on the surface of the plasma membrane of the ovum and embryo during fertilisation and cleavage in preparation for implantation. Glycoproteins are thought to be one of the factors in cell attachment. Thus, we investigated the changes in glycoprotein expression on the cell surface membrane of the mouse embryo by using lectins. Among seven types of lectin (ConA, WGA, UEA-I, MPA, LCA, DBA and PNA), the fluorescent intensities of ConA and WGA markedly increased from unfertilised ova to blastocysts. By quantitative analysis using immuno-scanning electron microscopy, the numbers of ConA-gold particles were small until 4-cell cleavage, but increased significantly at the blastocyst stage. In contrast, an increased number of WGA-gold particles was detected even at the 4-cell stage, and this increase continued to the blastocyst stage. From the above observations, we conclude that the numbers of sugar chains bound to both ConA and WGA increases with blastocyst formation and earlier expression is observed with WGA. The present study clearly shows that glycoproteins on the cell membrane surface of the mouse embryo quantitatively increase at the time of implantation, and the possibility has been indicated that glycoproteins are involved in intercellular recognition and adhesion between the embryo and endometrial epithelium.

A 58 kDa excretory–secretory product (ESP) of Giardia lamblia has been characterized. The ESP was purified over 508-fold by a combination of ammonium sulphate precipitation and sequential chromatography on affinity matrix and a gel filtration column. The homogeneity of the purified protein was established by sodium dodecyl sulphate polyacrylamide gel electrophoresis (Mr, 58 kDa) and analytical isoelectrophoresis (pI 4·75). The purified protein was recognized by the pooled sera of G. lamblia-positive patients as well as an antiserum raised against crude Giardia extract, thus indicating it to be an immunodominant parasite product. The ESP was found to agglutinate rabbit erythrocytes. The haemagglutinating activity of this protein was inhibited strongly by thyroglobulin, fetuin, asialofetuin and monosialoganglioside but not by simple sugars. The purified protein was characterized immunochemically and was found to be heat stable as well as protease sensitive. Lectin-binding studies of the purified ESP and its sensitivity to periodic-acid silver staining as well as to metaperiodate treatment clearly indicated its glycoprotein nature. The major localization site of the ESP was found to be on the surface of the parasite as revealed by flow cytometric analysis. Further, this glycoprotein induced fluid accumulation in ligated rabbit ileal loops and revealed a positive skin permeability reaction in the rabbit.

A lectin with hemagglutinating activity has been isolated from an aqueous extract of the symbiotic phenotype of Dictyonema glabratum and its cyanobacterial photobiont Scytonema sp. The purified lectin had a pI of 6.8 and its molecular mass was investigated by electrospray ionization mass spectrometry, gel filtration and SDS-PAGE, which indicated its native conformation as a dimer formed by two identical subunits of 16540 Da. The lectin is a glycoprotein with a low degree of glycosylation, containing galactose, xylose, glucose and mannose as neutral monosaccharides, in addition to glucosamine, which could indicate both N- and O-linkages. Amino acid analysis showed the predominance of nonpolar residues such as phenylalanine. Agglutination of human erythrocytes required divalent cations, which is affected by addition of EDTA. The lectin was more stable at 30 °C or less for at least 1 h and between pH 5.0 and 7.0. Among the various compounds tested for hemagglutination inhibition, N-acetylgalactosamine was the most active. The potential role of this lectin in recognition of the compatible cyanobacterial photobiont is discussed.

The intermolecular contact regions between monomers of the homodimeric DNA binding protein ParR and the interaction between the glycoproteins CD28 and CD80 were investigated using a strategy that combined chemical cross-linking with differential MALDI-MS analyses. ParR dimers were modified in vitro with the thiol-cleavable cross-linker 3,3′-dithio-bis(succinimidylproprionate) (DTSSP), proteolytically digested with trypsin and analyzed by MALDI-MS peptide mapping. Comparison of the peptide maps obtained from digested cross-linked ParR dimers in the presence and absence of a thiol reagent strongly supported a “head-to-tail” arrangement of the monomers in the dimeric complex. Glycoprotein fusion constructs CD28-IgG and CD80-Fab were cross-linked in vitro by DTSSP, characterized by nonreducing SDS-PAGE, digested in situ with trypsin and analyzed by MALDI-MS peptide mapping (± thiol reagent). The data revealed the presence of an intermolecular cross-link between the receptor regions of the glycoprotein constructs, as well as a number of unexpected but nonetheless specific interactions between the fusion domains of CD28-IgG and the receptor domain of CD80-Fab. The strategy of chemical cross-linking combined with differential MALDI-MS peptide mapping (± thiol reagent) enabled localization of the interface region(s) of the complexes studied and clearly demonstrates the utility of such an approach to obtain structural information on interacting noncovalent complexes.