Influence of fetuin and hyaluronan on the post-thaw quality and fertilizing ability of Holstein bull semen.

It was determined that fetuin and hyaluronan supplementation did not provide any significant effect on the post-thaw subjective and CASA motility percentages and sperm motion characteristics, in comparison to the controls (P>0.05). Sperm acrosome and total abnormalities were similar in all groups (P>0.05). Groups M (hyaluronan+fetuin) and H (hyaluronan) displayed a higher rate of sperm membrane integrity, compared to that of Group C (control) (P<0.01). According to the results of the comet assay, the lowest percentage of sperm with damaged DNA was achieved in Group H, when compared to all of the experimental groups (P<0.01). Furthermore, all of the additives resulted in a lower rate of sperm with damaged DNA than that of the controls, and thus, reduced DNA damage (P<0.01). For pregnancy rates, there were no significant differences between the extender groups (P>0.05). MDA formation was found to be lower in Groups M and F (P<0.01). In Group M, SOD activity was determined to have significantly increased (23.61±5.62 U/ml) compared to the other groups (P<0.01). All experimental groups had a GSH-Px activity higher than that of the control group (P<0.01).

Antioxidative effects of cysteamine, hyaluronan and fetuin on post-thaw semen quality, DNA integrity and oxidative stress parameters in the Brown Swiss bull.

The aim of this study was to compare the effectiveness of antioxidants including cysteamine (2.5, 7.5 mm), hyaluronan (0.25, 1 mg ml(-1) ) and fetuin (5, 10 mg ml(-1) ) in the freezing of Brown Swiss bull semen. The best percentages of CASA motilities were achieved with 10 mg ml(-1) of fetuin and 2.5 mm of cysteamine. For sperm morphology, 10 mg ml(-1) of fetuin and 2.5 mm of cysteamine had better protective effects (P < 0.001). The results of hypo-osmotic swelling test showed that the percentage values of membrane integrity in all the groups, excluding that supplemented with 5 mg ml(-1) of fetuin, were higher than those of the control group (P < 0.001). Results obtained for the DNA damage of sperm cells demonstrated that 0.25 mg ml(-1) of hyaluronan, and 2.5 and 7.5 mm of cysteamine led to lower rates of spermatozoa with damaged DNA, compared with the control group (P < 0.001). The maintenance of superoxide dismutase and glutathione peroxidase antioxidant activities following freeze-thawing with 2.5 and 7.5 mm of cysteamine and 10 mg ml(-1) of fetuin was demonstrated to be at a higher level in comparison with the control group (P < 0.001). Malondialdehyde formation was found to be lower in the groups supplemented with 0.25 mg ml(-1) of hyaluronan and 7.5 mm of cysteamine after the freeze-thawing process (P < 0.001).

Implications of the E-selectin S128R mutation for drug discovery.

The C-type lectin E-selectin mediates the rolling of circulating leukocytes on vascular endothelial cells during the inflammatory process. In numerous studies, the S128R mutation of the E-selectin was associated with cardiovascular and autoimmune diseases. There is evidence that the S128R E-selectin mutation leads to a loss in ligand specificity, thus increasing leukocyte recruitment. Apart from the natural tetrasaccharide ligand sialyl Lewis(x) (sLe(x)), it has previously been proposed that non-fucosylated carbohydrates also bind to S128R E-selectin. To evaluate the therapeutic potential of the antagonism of the E-selectin mutant, ligand specificity was reinvestigated on a molecular basis. We determined the ligand specificity of wild-type and S128R E-selectin in a target-based competitive assay, a glycan array screen and cell-based binding assays under static and flow conditions. Regarding ligand-specificity, the binding properties of S128R E-selectin were identical to those of wt E-selectin, i.e., no mutant-specific binding of 3'-sialyl-N-acetyllactosamine, heparin, fetuin and K562 cells was observed. Additionally, the binding affinities of glycomimetic E-selectin antagonists were identical for wt and S128R E-selectin. Overall, the previous reports on carbohydrate ligand promiscuity of S128R E-selectin could not be confirmed.

Role of sialic acids in feline enteric coronavirus infections.

To initiate infections, many coronaviruses use sialic acids, either as receptor determinants or as attachment factors helping the virus find its receptor underneath the heavily glycosylated mucus layer. In the present study, the role of sialic acids in serotype I feline enteric coronavirus (FECV) infections was studied in feline intestinal epithelial cell cultures. Treatment of cells with neuraminidase (NA) enhanced infection efficiency, showing that terminal sialic acid residues on the cell surface were not receptor determinants and even hampered efficient virus-receptor engagement. Knowing that NA treatment of coronaviruses can unmask viral sialic acid binding activity, replication of untreated and NA-treated viruses was compared, showing that NA treatment of the virus enhanced infectivity in untreated cells, but was detrimental in NA-treated cells. By using sialylated compounds as competitive inhibitors, it was demonstrated that sialyllactose (2,6-α-linked over 2,3-α-linked) notably reduced infectivity of NA-treated viruses, whereas bovine submaxillary mucin inhibited both treated and untreated viruses. In desialylated cells, however, viruses were less prone to competitive inhibition with sialylated compounds. In conclusion, this study demonstrated that FECV had a sialic acid binding capacity, which was partially masked by virus-associated sialic acids, and that attachment to sialylated compounds could facilitate enterocyte infections. However, sialic acid binding was not a prerequisite for the initiation of infection and virus-receptor engagement was even more efficient after desialylation of cells, indicating that FECV requires sialidases for efficient enterocyte infections.

Occurrence of novel Cu(2+)-dependent sialic acid-specific lectin, on the outer surface of mature caprine spermatozoa.

Effects of several bivalent metal ions on the autoagglutination event in mature caprine epididymal sperm cells have been investigated using a chemically defined medium. This study demonstrates for the first time that Copper (Cu(2+)) ion (300 µM) has high specificity for autoagglutination of mature cauda-epididymal sperm. Head-to-head interaction of the male gametes is responsible for this event. Studies on the effect of various sugars reveal that the autoagglutinated cells can be dissociated specifically with neutralized sialic acid (50 mM), which also inhibits the sperm cell autoagglutination phenomenon. Blood serum protein fetuin, that contains terminal sialic acid residue, showed high efficacy for inhibiting this autoagglutination event at 4 µM concentration. However, asialofetuin is not capable of inhibiting this Cu(2+)-dependent cellular event. Mature sperm cells bound with caprine erythrocytes at their head region in presence of Cu(2+) ion. The purified sperm membrane fraction isolated by aqueous two phase polymer method showed high efficacy to agglutinate erythrocytes. These sperm-erythrocyte interactions as well as sperm membrane induced haemagglutination were strongly blocked by neutralized sialic acid (50 mM). The results confirm the occurrence of unique Cu(2+) dependent, sialic acid-specific lectin on the outer surface of a mammalian cell using caprine sperm as the model. The observed Cu(2+)-mediated cellular autoagglutination is caused by the interaction of the cell surface lectin with the lectin receptor on the surface of the neighboring homologous cell.

Hepatocyte-targeting gene delivery using a lipoplex composed of galactose-modified aromatic lipid synthesized with click chemistry.

Highly efficient drug carriers targeting hepatocyte is needed for treatment for liver diseases such as liver cirrhosis and virus infections. Galactose or N-acetylgalactosamine is known to be recognized and incorporated into the cells through asialoglycoprotein receptor (ASGPR) that is exclusively expressed on hepatocyte and hepatoma. In this study, we synthesized a galactose-modified lipid with aromatic ring with click chemistry. To make a complex with DNA, termed 'lipoplex', we prepared a binary micelle composed of cationic lipid; dioleoyltrimethylammoniumpropane (DOTAP) and galactose-modified lipid (D/Gal). We prepared lipoplex from plasmid DNA (pDNA) and D/Gal and examined the cell specificity and transfection efficiency. The lipoplex was able to interact with ASGPR immobilized on gold substrate in the quartz-crystal microbalance (QCM) sensor cell. The lipoplex induced high gene expression to HepG2 cells, a human hepatocellular carcinoma cell line, but not to A549 cells, a human alveolar adenocarcinoma cell line. The treatment with asialofetuin, which is a ligand for ASGPR and would work as a competitive inhibitor, before addition of the lipoplexes decreased the expression to HepG2 cells. These results indicate that D/Gal lipoplex was incorporated into HepG2 cells preferentially through ASGPR and the uptake was caused by galactose specific receptor. This delivery system to hepatocytes may overcome the problems for gene therapy and be used for treatment of hepatitis and hepatic cirrhosis.

Scavenger receptor-mediated uptake of cell-penetrating peptide nanocomplexes with oligonucleotides.

Cell-penetrating peptides (CPPs) are short cationic peptides that penetrate cells by interacting with the negatively charged plasma membrane; however, the detailed uptake mechanism is not clear. In contrary to the conventional mode of action of CPPs, we show here that a CPP, PepFect14 (PF14), forms negatively charged nanocomplexes with oligonucleotides and their uptake is mediated by class-A scavenger receptors (SCARAs). Specific inhibitory ligands of SCARAs, such as fucoidin, polyinosinic acid, and dextran sulfate, totally inhibit the activity of PF14-oligonucleotide nanocomplexes in the HeLa pLuc705 splice-correction cell model, while nonspecific, chemically related molecules do not. Furthermore, RNA interference (RNAi) knockdown of SCARA subtypes (SCARA3 and SCARA5) that are expressed in this cell line led to a significant reduction of the activity to <50%. In line with this, immunostaining shows prevalent colocalization of the nanocomplexes with the receptors, and electron microscopy images show no binding or internalization of the nanocomplexes in the presence of the inhibitory ligands. Interestingly, naked oligonucleotides also colocalize with SCARAs when used at high concentrations. These results demonstrate the involvement of SCARA3 and SCARA5 in the uptake of PF14-oligonucleotide nanocomplexes and suggest for the first time that some CPP-based systems function through scavenger receptors, which could yield novel possibilities to understand and improve the transfection by CPPs.

Jatropha curcas hemagglutinin is similar to a 2S albumin allergen from the same source and has unique sugar affinities.

We have previously reported the purification and preliminary X-ray characterization of a hemagglutinin from the seeds of Jatropha curcas and, with the detailed sequencing information available now, we find that it is similar to a 2S albumin allergen isolated from the same source. Through a search of Jatropha genome database (http://www.kazusa.or.jp/jatropha/), we map it to the sequence id JcCA0234191 (now referred to as Jcr4S00619.70 in the new version, release 4.5) which has a conserved alpha amylase inhibitor/seed storage protein domain found in the 2S albumin allergens. The putative sequence of the small and large chains of the protein is assigned and the total mass of the two subunits matches with the intact mass 10 kDa determined through MALDI. The protein retains hemagglutination activity between pH 6-9 and up to 60 °C on heat treatment and its hemagglutination activity is inhibited by sialic acid and fetuin. Bioinformatics studies show that the isolated protein sequence clusters in close association with a 2S albumin from Ricinus communis in phylogeny analysis and has a conservation of the characteristic four disulfide linkage pattern. Hemagglutinins and lectins are known to have allergenic effects through their interaction with immunoglobulin E and histamine release and earlier studies have shown that this interaction can be inhibited by lectin-specific sugars. We hope this report bridges the plant allergens and hemagglutinins further for exploring possible mediation of allergenic activity through sialic acid and complex sugar interactions and generates further interest in the area.

Purification of a lectin from Arisaema erubescens (Wall.) Schott and its pro-inflammatory effects.

The monocot lectin from the tubers of Arisaema erubescens (Wall.) Schott has been purified by consecutive hydrophobic chromatography and ion exchange chromatography methods. The molecular weight of this A. erubescens lectin (AEL) was determined to be about 12 kDa by high performance liquid chromatography (HPLC) and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) methods. AEL could agglutinate rabbit erythrocytes. The haemagglutination activity of AEL was only inhibited by asialofetuin, while monosaccharide did not react. Rat paw edema and neutrophil migration models were used to investigate the pro-inflammatory activity of AEL. AEL (100 and 200 µg/paw) could induce significant rat paw edema. In addition, AEL (100, 200 and 300 µg/mL/cavity) could induce significant and dose-dependent neutrophil migration in the rat peritoneal cavities. Besides, AEL at doses ranging from 100 to 300 µg/mL/cavity could significantly increase the concentration of nitric oxide (NO), prostaglandin E(2 )(PGE(2)) and tumor necrosis factor alpha (TNF-α) in peritoneal fluid. As compared with control animals, 75% depletion in the number of resident cells following peritoneal lavage did not reduce the AEL-induced neutrophil migration. However, pre-treatment with 3% thioglycollate which increased the peritoneal macrophage population by 201%, enhanced the neutrophil migration induced by AEL (200 µg/mL/cavity) (p < 0.05). Reduction of peritoneal mast cell population by chronic treatment of rat peritoneal cavities with compound 48/80 (N-methyl-p-methoxyphenethylamine with formaldehyde) did not modify AEL-induced neutrophil migration. The results provided the basis for identifying the toxic components of A. erubescens and AEL could be a new useful tool for pro-inflammatory research.

Ovarian tissue culture in the presence of VEGF and fetuin stimulates follicle growth and steroidogenesis.

Ovarian tissue cryopreservation together with follicle culture provides a promising technique for fertility preservation in cancer patients. The study aimed to evaluate follicle parameters in a culture medium supplemented with VEGFA165 and/or fetuin. Vitrified-warmed ovarian cortical pieces were divided randomly into four culture groups consisting of basic culture medium (control), and the basic culture medium supplemented with VEGFA165, fetuin or both. After six days of culture, we evaluated the following: percentage of resting, primary and secondary growing follicles; survival rate; steroid hormones production; levels of reactive oxygen species, lipid peroxidation and total antioxidant capacity; and developmental and antioxidant gene expression. The addition of VEGFA165 alone or in combination with fetuin to the culture medium caused resting follicle activation and increased the number of growing follicles. In the VEGFA165 group, we found a significant increase in the concentrations of 17ß-estradiol at day 6 and progesterone from 4th day of the culture period. In the VEGFA165 + fetuin group, the concentration of 17ß-estradiol rose at day 4 of the culture period. The levels of BMP15, GDF9 and INHB mRNAs were increased in all treated groups. In the fetuin and fetuin + VEGFA165 groups, we observed a high level of total antioxidant capacity and expression of SOD1 and CAT genes, low reactive oxygen species and lipid peroxidation levels and increased number of viable follicles. In conclusion, the present study provides useful evidence that supplementation of culture medium with VEGFA165 + fetuin leads to primordial follicle activation and development and increased percentage of healthy secondary growing follicles.

Lectin from inflorescences of ornamental crop Alpinia purpurata acts on immune cells to promote Th1 and Th17 responses, nitric oxide release, and lymphocyte activation.

Alpinia purpurata is an ornamental crop known as a source of bioactive molecules. This is the first study to report isolation of a lectin (carbohydrate-binding protein) from A. purpurata inflorescences (ApuL). The immunomodulatory potential of ApuL was evaluated by investigating its effects on the production of cytokines and release of nitric oxide by human peripheral blood mononuclear cells (PBMCs). In addition, the differentiation and activation of lymphocytes treated with ApuL was evaluated by immunophenotyping assays. ApuL is an acidic and oligomeric protein with native molecular mass of 34kDa. The hemagglutinating activity (HA) of ApuL was inhibited by the glycoproteins fetuin and ovalbumin, was resistant to heating at 100°C and stimulated in the presence of calcium and magnesium ions. ApuL showed highest HA at pH 7.5 but failed to agglutinate erythrocytes at pH 8.0 and 9.0. ApuL induced the release of cytokines belonging to Th1 (IFN-γ, TNF-α, and IL-6) and Th17 (IL-17A) profiles as well as of nitric oxide, stimulating a pro-inflammatory environment. Moreover, ApuL also stimulated the production of IL-10, an anti-inflammatory cytokine with regulatory role. Incubation with lectin resulted in differentiation and activation of both T CD8+ and CD4+ subsets of lymphocytes, as evident from the expression of the CD28 costimulatory molecule. In conclusion, A. purpurata inflorescence is a source of an immunomodulatory lectin with potential immunoregulatory application, thereby adding biotechnological value to this ornamental crop.

Remineralization of demineralized bone matrix (DBM) via alternating solution immersion (ASI).

In order to achieve successful clinical outcomes, biomaterials used for bone grafts must possess a number of traits including biocompatibility and osteoconductivity. These materials must also demonstrate appropriate mechanical stability to withstand handling as well as support potentially significant stresses at the implant site. Synthetic and natural polymer scaffolds used for bone tissue engineering (BTE) often lack necessary mechanical properties. Our goal was to internally mineralize natural collagenous matrix, thereby increasing mechanical properties of the material to useful levels. Published methods for intrafibrillar collagen mineralization were applied to clinically relevant-sized constructs but did not successfully deposit mineral in the interior of the constructs. To address this limitation, we developed a new technique for the remineralization of demineralized bone matrix (DBM) based on alternating solution immersion, or ASI. Mineral was removed from equine bone specimens, leaving behind a demineralized bone matrix (DBM). This matrix provides a framework for the nucleation and growth of a replacement mineral phase. Plain film radiography and microcomputed tomography (microCT) indicated accumulation of mineral within the DBM, and mechanical testing (3 point bending and compression) revealed a significant increase in stiffness between the DBM and the remineralized bone matrix (RBM). We believe this remineralization process will be useful in the preparation of stiff and strong allografts for clinical application.

Primary porcine Kupffer cell phagocytosis of human platelets involves the CD18 receptor.

BACKGROUND: Hepatic failure has been treated successfully with clinical extracorporeal perfusions of porcine livers. However, dog-to-pig and pig-to-baboon liver xenotransplant models have resulted in severe bleeding secondary to liver xenograft-induced thrombocytopenia. Kupffer cells (KC) are abundant phagocytic cells in the liver. KC express the CD11b/CD18 receptor, which has been implicated in chilled platelet binding and phagocytosis through interaction with platelet surface proteins and carbohydrates. We sought to identify the role of KC CD18 in liver xenograft-induced thrombocytopenia. METHODS: Primary pig KC were characterized by flow cytometry, immunoblots, and quantitative polymerase chain reaction. Pig KC were used in inhibition assays with fluorescently labeled human platelets. The CD18 receptor was targeted for siRNA knockdown. RESULTS: Domestic and α1,3-galactosyltransferase double knockout porcine KC cultures were approximately 92% positive for CD18 as detected by quantitative polymerase chain reaction and flow cytometry. Use of CD18 blocking antibodies resulted in reduction of human platelet binding and phagocytosis. Additionally, asialofetuin, not fetuin, inhibited platelet phagocytosis suggesting the involvement of an oligosaccharide-binding site. Furthermore, reduced CD18 expression by siRNA resulted in decreased human platelet binding. CONCLUSIONS: Our data suggest that primary pig KC bind and phagocytose human platelets with involvement of CD18. Further understanding and modification of CD18 expression in pigs may result in a liver xenograft with reduced thrombocytopenic effects, which could be used as a bridge to allogeneic liver transplantation.