Leonardo Ermini

Protein profile of capacitated versus ejaculated human sperm.

Freshly ejaculated sperm acquire the fertilizing potential by a continuing process that occurs during sperm transport through the female genital tract, and it is physiologically not complete until the spermatozoon reaches the oocyte. The process termed capacitation can be mimicked in vitro by using appropriate capacitation media. Despite its importance, the molecular mechanisms underlying capacitation are poorly understood. This work deals with a proteomic approach to the analysis of protein profile variations in human normospermic samples as a consequence of three hours in vitro capacitation. 2DE gels were produced per freshly ejaculated sperm and per capacitated sperm and several quantitative and qualitative significant variations were found. Among the MS obtained identifications, proteins with a significant decrease after capacitation were found to be involved in protein fate, metabolism, and flagellar organization; on the contrary, increasing proteins were found to be related to cellular stress. Interestingly, the detected flagellar organization proteins decreased during capacitation whereas their corresponding fragments increased. A swim-up selected and three-hour capacitated sperm subpopulation has also been resolved by 2DE, and its synthetic gel has been analyzed for the variations observed in the entire sperm population. An immunofluorescence analysis of this sperm typology was carried out with antiactin and antitubulin antibodies.

Nestin Expression in Adult and Developing Human Kidney

Nestin is considered a marker of neurogenic and myogenic precursor cells. Its arrangement is regulated by cyclin-dependent kinase 5 (CDK5), which is expressed in murine podocytes. We investigated nestin expression in human adult and fetal kidney as well as CDK5 presence in adult human podocytes. Confocal microscopy demonstrated that adult glomeruli display nestin immunoreactivity in vimentin-expressing cells with the podocyte morphology and not in cells bearing the endothelial marker CD31. Glomerular nestin-positive cells were CDK5 immunoreactive as well. Western blotting of the intermediate filament-enriched cytoskeletal fraction and coimmunoprecipitation of nestin with anti-CDK5 antibodies confirmed these results. Nestin was also detected in developing glomeruli within immature podocytes and a few other cells. Confocal microscopy of experiments conducted with antibodies against nestin and endothelial markers demonstrated that endothelial cells belonging to capillaries invading the lower cleft of S-shaped bodies and the immature glomeruli were nestin immunoreactive. Similar experiments carried out with antibodies raised against nestin and α-smooth muscle actin showed that the first mesangial cells that populate the developing glomeruli expressed nestin. In conclusion, nestin is expressed in the human kidney from the first steps of glomerulogenesis within podocytes, mesangial, and endothelial cells. This expression, restricted to podocytes in mature glomeruli, appears associated with CDK5.

RNA‐mediated gene silencing of FUT1 and FUT2 influences expression and activities of bovine and human fucosylated nucleolin and inhibits cell adhesion and proliferation

In a previous article, we demonstrated the existence of fucosyl‐containing O‐glycans forms of nucleolin in bovine post‐capillary venular endothelial cells (CVEC) and malignant cultured human A431 cells. The tool for this discovery was an antibody found to interact strongly and exclusively with nucleolin in total protein extracts. The antibody was originally raised against a mollusc glycoprotein and was demonstrated to be directed against its O‐glycans, recently found to belong prevalently to the blood group H‐antigen type with fucose linked in alpha1, 2 to galactose. Here, we show that si‐RNA induced down‐regulation of the expression of FUT1 and FUT2, the fucosyltransferases required for the biosynthesis of the terminal glycan motif Fucα‐2‐Galβ‐R, reduced expression of the fucosylated nucleolin glycoforms and their exposure at the cell surface in CVEC. Treatment of the cells with FUT1/2 siRNA also reduced their ability to bind and internalize endostatin and their adhesion efficiency and inhibited cell growth. Expression of FUT1, FUT2, and FUT6 was also analyzed in serum‐stimulated versus serum‐starved cells and in cells treated with FUT1 and FUT2 siRNA. A reduced expression of fucosylated nucleolin and inhibition of cell growth by suppressing FUT1/2 expression was also tested and shown to be exhibited in human A431 cells. J. Cell. Biochem. 111: 229–238, 2010.

Rabbit Tonsil-associated M-cells Express Cytokeratin 20 and Take Up Particulate Antigen

M-cells are believed to play a pivotal role in initiation of the immune response. These cells, located in the epithelia that overlie mucosal lymphoid follicles, are responsible for the active uptake of particulate antigens and for their translocation to the underlying lymphoid tissue. The identification of reliable markers for M-cells is therefore extremely important for the study of the initial steps that lead to the immune response. For this purpose, we studied cytokeratin 20 (CK20) expression in the epithelium of rabbit palatine tonsils by immunofluorescence, confocal microscopy, and Western blotting. CK20+ cells were observed in all rabbit palatine tonsils examined. By Western blotting, one CK20-immunoreactive band was identified at 46 kD on samples of proteins from the intermediate filament-enriched cytoskeletal fraction of tonsil epithelium. Double labeling of CK20+ cells with cell-specific markers confirmed that such cells were actually M-cells. Moreover, CK20+ M-cells displayed a mature phenotype (they formed pockets harboring lymphoid cells) and were functionally competent because they could take up particulate antigens from the pharyngeal lumen. We conclude that CK20 is an M-cell marker for rabbit palatine tonsils. Moreover, we can hypothesize the use of M-cells as a possible site for antigen delivery of particle-based vaccines.

An Appraisal of Intermediate Filament Expression in Adult and Developing Pancreas: Vimentin Is Expressed in α Cells of Rat and Mouse Embryos

Intermediate filaments are frequently used in studies of developmental biology as markers of cell differentiation. To assess whether they can be useful to identify differentiating pancreatic endocrine cells, we examined the pattern of expression of nestin, cyto-keratin 20, and vimentin on acetone-fixed cryosections of rat adult and developing pancreas. We also studied vimentin expression in mouse embryonic pancreas at E19. Cytokeratin 20 was found in all pancreatic epithelial cell lineages during the entire development of the rat gland and in the adult animals. Under our experimental conditions, therefore, cytokeratin 20 is not an exclusive marker of rat duct cells. Nestin was detected exclusively in stromal cells either in the adult or developing rat pancreas. Vimentin was observed within cells located in the primitive ducts of rat pancreas starting from E12.5. Their number rapidly increased, reaching its highest level in newborn animals. Vimentin was also spotted in α cells starting from E12.5 but disappeared soon after birth, likely identifying immature or recently differentiated α cells. In addition, vimentin was observed in duct and α cells of mouse developing pancreas showing that its expression in such cells is not an event restricted to the rat. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Low concentrations of Bisphenol A and para-Nonylphenol affect extravillous pathway of human trophoblast cells

Bisphenol A (BPA) and para-Nonylphenol (p-NP) are chemicals of industrial origin which may influence human reproductive health. The effects of these substances in the prenatal life is an important topic that is receiving greater attention in the developed countries. In this study, human trophoblast cells HTR-8/SVneo were exposed to BPA and p-NP (1 × 10(-15), 1 × 10(-13), 1 × 10(-11), 1 × 10(-9) and 1 × 10(-7) M) and incubated for 24, 48 and/or 72 h then, examined for the main physiological processes which characterize the extravillous trophoblast. Cell proliferation showed no changes while the processes of cell migration and invasion were both reduced by BPA and p-NP. For each chemical, the activity was higher at lower concentrations with a maximum activity between 1 × 10(-13) and 1 × 10(-11) M (p < 0.05 for 1 × 10(-9) and p < 0.001 for 1 × 10(-11) M). Co-culture studies with human umbilical cord endothelial cells (HUVEC) revealed that trophoblast/endothelial interaction was significantly reduced by p-NP at 1 × 10(-11) M. Moreover, both chemicals were inducing differentiation of HTR-8/SVneo toward polyploidy by the process of endoreduplication. The estrogen-receptor antagonist ICI significantly reduced p-NP action, while it had no effect on BPA treated cells. In conclusion, p-NP and BPA act on trophoblast cells altering key physiological processes in placenta development. The exact mechanism of action of the chemicals in human trophoblast still needs to be clarified.

Formulation of liposomes functionalized with Lotus lectin and effective in targeting highly proliferative cells

BACKGROUND Liposomes, used to improve the therapeutic index of new and established drugs, have advanced with the insertion of active targeting. The lectin from Lotus tetragonolobus (LTL), which binds glycans containing alpha-1,2-linked fucose, reveals surface regionalized glycoepitopes in highly proliferative cells not detectable in normally growing cells. In contrast, other lectins localize the corresponding glycoepitopes all over the cell surface. LTL also proved able to penetrate the cells by an unconventional uptake mechanism. METHODS We used confocal laser microscopy to detect and localize LTL-positive glycoepitopes and lectin uptake in two cancer cell lines. We then constructed doxorubicin-loaded liposomes functionalized with LTL. Intracellular delivery of the drug was determined in vitro and in vivo by confocal and electron microscopy. RESULTS We confirmed the specific localization of Lotus binding sites and the lectin uptake mechanism in the two cell lines and determined that LTL-functionalized liposomes loaded with doxorubicin greatly increased intracellular delivery of the drug, compared to unmodified doxorubicin-loaded liposomes. The LTL-Dox-L mechanism of entry and drug delivery was different to that of Dox-L and other liposomal preparations. LTL-Dox-L entered the cells one by one in tiny tubules that never fused with lysosomes. LTL-Dox-L injected in mice with melanoma specifically delivered loaded Dox to the cytoplasm of tumor cells. CONCLUSIONS Liposome functionalization with LTL promises to broaden the therapeutic potential of liposomal doxorubicin treatment, decreasing non-specific toxicity. GENERAL SIGNIFICANCE Doxorubicin-LTL functionalized liposomes promise to be useful in the development of new cancer chemotherapy protocols.

Oxygen governs Galβ1–3GalNAc epitope in human placenta

It is becoming increasingly apparent that the dynamics of glycans reflect the physiological state of cells involved in several cell functions including growth, response to signal molecules, migration, as well as adhesion to, interaction with, and recognition of other cells. The presence of glycoconjugates in human placenta suggests their major role in maternal-fetal exchanges, intercellular adhesion, cellular metabolism, and villous vessel branching. Although several studies have described glycoconjugate distribution in the human placenta descriptions of their physiological function and control mechanisms during placental development are lacking. In this study we investigated the developmental distribution and regulation of placental core 1 O- and N-glycans focusing on early and late first trimester human pregnancy. To define the control mechanisms of the oligosaccharide chains during early placentation process, chorionic villous explants and human trophoblast cell lines were exposed to various oxygen levels. We found that oxygen tension regulates changes in core-1 O-glycan (the disaccharide Galβ1-3GalNAc) epitope expression levels. Moreover, by double affinity chromatography and subsequent analysis with mass spectrometry, we identified in the heat shock protein 90-α (HSP90α) a good candidate as carrier of the Galβ1-3GalNAc epitope at low oxygen tension. Our results support a fundamental role of oxygen tension in modulating glycosylation of proteins during placental development.

Identification of a novel, alpha2-fucosylation-dependent uptake system in highly proliferative cells

In this paper we describe a new structure present in highly proliferative cells and absent in cells with normal growth potential. We used cultured bovine venular endothelial cells (CVEC) as examples of high proliferation, and dermal fibroblasts of a primary culture as examples of normal proliferation. The structure, consisting of tubules radiating from the nuclear region to the tips of cell protrusions, was revealed by its strong positivity to the fucose-binding lectin from Lotus (LTL) that prefers glycans with alpha-1,2-linked fucose. Another fucose-binding lectin that prefers glycans with alpha-1,6-linked fucose was instead found to localize glycans exclusively in Golgi complexes. LTL binding sites were also found at the surface of CVEC in a restricted region close to the nucleus. The role of alpha-1,2-linked fucose in forming or maintaining the tubules was confirmed by the fact that down-regulation of the fucosyltransferases FUT1 and FUT2 resulted in disappearance of the tubular structure. LTL also proved able to penetrate the cells through the tubular structures up to the nuclear region and to inhibit proliferation. Endostatin was also found to massively penetrate the cells in the tubular structures in control cells but not in FUT1/2 depleted cells. In cells of a first passage primary culture of dermal fibroblasts the tubular LTL-positive structure was absent as well as the LTL-positive sites at the external surface, and both fucose-binding lectins were found to exclusively localize glycans in Golgi complexes. Tubules were again found progressively in fibroblasts derived from repeated passages, where faster growing cells predominate. Disappearance of LTL-positivity in Golgi complexes paralleled appearance of LTL-positive tubules. The role of Golgi complexes in forming the tubules is discussed.

Expression of CD52 mRNA in the rat embryo

CD52 is a leukocyte differentiation antigen first discovered in humans as expressed on the surface of lymphocytes, monocytes and eosinophils. The human CD52 is found on chromosome 1, and two alleles are both known to be reasonably common. A closely homologous gene has been identified in the cynomologous monkey and related genes have been found in mouse, rat and dog. The role of CD52 in lymphocyte is still unclear but the anti‐CD52 antibodies named CAMPATH‐1 antibodies are largely used for therapy where depletion of lymphocytes is required. In the past expression of the antigen on progenitors of leukocytes in bone marrow had been excluded, but recent work indicates CD52 is highly expressed on cells with colony‐forming and NOD/SCID (non‐obese diabetic‐severe combined immunodeficiency)‐engrafting capacities, both at the mRNA and membrane protein level. We have investigated CD52 expression during development in rat embryos by in situ hybridization. We report here that the antigen is highly expressed in the liver that is the major organ where multipotent hematopietic stem cells differentiate but also in the splancnopleuric mesoderm, at early stages of embryo differentiation, where hematopietic stem cells are suggested to arise. CD52+ cells were found in areas active in vasculogenesis at early embryo stages and in the walls of the vessels in the liver at mid gestation. CD52+ cells were also found to emerge among c‐Kit positive cells.