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Vitelline coat of Unio elongatulus: II. Biochemical properties of the 220- and 180-kD components
No full textIn a previous study we found that two glycoproteins with apparent molecular weights of 220 kD and 180 kD account for 80-90% of the material dissolved from the vitelline coat of the egg of the bivalve mollusk, Unio elongatulus (Focarelli and Rosati, 1993: Mol Reprod Dev 35:44-51). In this study we isolated and purified these glycoproteins by electroelution. The two proteins differ in many respects: the 180-kD molecule is acidic in nature and highly heterogeneous, whereas the 220-kD protein is neutral and less heterogeneous. Both seem to have O- and N-linked oligosaccharide chains. The 180-kD protein contains 13-16% carbohydrate, whereas the 220-kD molecular contains only 7-8%. Amino acid analysis and peptide mapping also show that each protein represents a unique polypeptide chain
'A role for mitochondrial respiration in human sperm hyperactivation as determined by a polarographic assay.
No full textA fucose-containing O-glycoepitope on bovine and human nucleolin
No full textIn this paper, we demonstrate the existence and localization
of fucosyl-containing O-glycoforms of nucleolin in cultured
bovine endothelial cells (CVEC) and malignant cultured
human A431 cells. The tool for this discovery was an
antibody raised against gp273, a glycoprotein ligand for the
sperm–egg interaction in the mollusc bivalve Unio elongatulus.
The function and immunological properties of gp273
mainly depend on clustered Lewis-like, fucose-containing
O-glycans. Here an anti-gp273 antibody was used to evaluate
whether glycoepitopes similar to those of gp273 are part
of potential ligands of selectins in endothelial cells.We found
that anti-gp273 strongly and exclusively interacted with a
110 kDa protein in CVEC and A431 tumor cells. After partial
purification, mass spectrometry identified the protein
as nucleolin. This was confirmed by comparing anti-gp273
and anti-nucleolin antibody immunoblotting after nucleolin
depletion.We confirmed that anti-gp273 binding to nuclear
and extranuclear nucleolin was against a fucose-containing
O-glycoepitope by immunoblot analysis of the protein after
chemically removing O-glycans and by lectin-blot analysis
of control and nucleolin-depleted samples. Using anti-gp273
IgG, we detected nucleolin on the plasma membrane and
cytoplasm. O-glycosylation may regulate the plethora of
functions in which nucleolin is involved
CD52, an intriguing antigen of the human sperm surface
No full textCD52 is the product of a single gene on chromosome 1, first characterized as a human leukocyte differentiation antigen expressed on the surface of lymphocytes and monocytes and then identified in the epithelial cells of epididymal corpus and cauda and in the deferent ducts. Sperm also have this antigen, acquiring it as they pass through the epididymis. The precursor of CD52 is 61 amino acids long, but the mature antigen, that serves as backbone for N-glycosylation and a final GPI-anchor, only contains 12 amino acids. O-linked oligosaccharide chains are also bound to certain glycoforms of this complex antigen. The first and most commonly used anti-CD52 monoclonal antibody, termed CAMPATH-1, generated in the immune system, interacts with the last three amino acids and the first part of GPI, and recognizes CD52 on leukocytes, epididymal cells and sperm. Other antibodies such as S19 and Mab H6-3C4 react only with CD52 carbohydrate epitopes specific to the reproductive system. We generated a polyclonal antibody that mainly recognizes CD52 glycoforms containing O-linked oligosaccharide chains. We called this antibody anti-gp20 because it was raised against a 20 kDa sialylglycoprotein, homologous to CD52, recovered as sole radiolabelled glycoprotein after radiolabelling surface sialic acid residues of capacitated human sperm. Anti-gp20 revealed that the O-glycan-containing glycoform in capacitated sperm differs from the other CD52 glycoforms in localization on the surface and microdomain segregation. This does not occur on leukocytes and freshly ejaculated sperm. Since this form specifically localizes in the equatorial region of sperm capacitated in vitro and anti-gp20 impairs sperm penetration of denuded hamster eggs, a role in sperm-egg fusion has been suggested for it. The capacity of CD52 to form a complex with semenogelin I recently indicated that the antigen also has a role in semen clot formation and liquefaction
Protein modification as oxidative stress marker in normal and pathological human seminal plasma
No full textObjective: Our study aims to assess the oxidative stress status of seminal plasma from normozoospermic, azoospermic, and leukocytospermic males, since abnormal sperm and leukocytes in human ejaculates are the main source of reactive oxygen species (ROS) which lead to oxidative damages. For this purpose we applied a biochemical approach to the assessment of the oxidative stress status by using twodimensional (2D) electrophoresis to check the level of protein oxidation after specific labeling of free thiol (-SH) groups. Methods: Seminal plasma samples from normal and pathological males were analyzed by a luminol-based chemiluminescent assay. The same samples after specific labeling of free -SH groups with 3-Nmaleimidopropionyl biocytin, were analyzed by 2D electrophoresis and computer-assisted semiquantitative determination of the amount of free -SH groups. Results: Using a standard chemiluminescence assay, we demonstrated a high, low and normal level of ROS, respectively, in seminal plasma from leukocytospermic, azoospermic, and normozoospermic subjects. By 2D electrophoresis and streptavidin blotting of specifically labeled free -SH groups of proteins, we detected in the same samples a higher level of oxidated -SH groups comparable between azoospermic and leukocytospermic samples, whereas a significantly higher level of free -SH groups was detected in normozoospermic subjects. Discussion: Our results demonstrated that a pathological oxidative stress status in seminal plasma may be revealed by the levels of the protein free -SH groups, both in the presence or absence of cells. © W.S. Maney & Son Ltd 2012
Identification and characterization of a 38 kDa glycoprotein functionally associated with mating activity of Paramecium primaurelia
No full textIn Paramecium primaurelia mating interactions take place immediately after mixing mating-competent cells of opposite mating types. The cells clump in clusters (mating reaction) and then separate in pairs. Previous results have
shown that sialic acid-containing glycoconjugates are present on the cell surface and are involved in mating-cell
pairing. In order to identify the sialic acid-containing glycoprotein(s), we first metabolically radiolabelled non-mating competent cells with D-[6-3H]galactose, and then analyzed the radiolabelled proteins by anion exchange
chromatography. We characterized a 38 kDa (gp38) sialic acid-containing glycoprotein and raised the corresponding
polyclonal antibody by means of which we localized the antigen at the level of the oral region of non-mating competent cells and on the ciliary surface of mating-competent cells. Immunoblot analysis of the ciliary protein
fraction showed that the anti-gp38 serum interacted with a 38 kDa protein in both mating types I and II cells. We also
demonstrated the functional activity of gp38 in the mating reaction by means of anti-gp38 antibody competition
assays
Protein pathways working in human follicular fluid: the future for tailored IVF?
Get PDFThe human follicular fluid (HFF) contains molecules and proteins that may affect follicle growth, oocyte maturation and competence acquiring. Despite the numerous studies, an integrated broad overview on biomolecular and patho/physiological processes that are proved or supposed to take place in HFF during folliculogenesis and oocyte development is still missing. In this review we report, for the first time, all the proteins unambiguously detected in HFF and, applying DAVID (Database for Annotation, Visualization and Integrated Discovery) and MetaCore bioinformatic resources, we shed new lights on their functional correlation, delineating protein patterns and pathways with reasonable potentialities for oocyte quality estimation in in vitro fertilisation (IVF) programs. Performing a rigorous PubMed search, we redacted a list of 617 unique proteins unambiguously-annotated as HFF components. Their functional processing suggested the occurrence in HFF of a tight and highly dynamic functional-network, which is balanced by specific effectors, primarily involved in extracellular matrix degradation and remodelling, inflammation and coagulation. Metalloproteinases, thrombin and vitamin-D-receptor/retinoid-X-receptor-alpha resulted as the main key factors in the nets and their differential activity may be indicative of ovarian health and oocyte quality. Despite future accurate clinical investigations are absolutely needed, the present analysis may provide a starting point for more accurate oocyte quality estimation and for defining personalised therapies in reproductive medicine
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
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