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1.
Histol Histopathol ; 24(8): 999-1007, 2009 08.
Article in English | MEDLINE | ID: mdl-19554507

ABSTRACT

This study evaluates the use of two fluorescein-labelled (FITC) plant lectins, Pisum sativum (edible pea) agglutinin (PSA) and Arachis hypogaea (peanut) agglutinin (PNA), in order to determine the most accurate and reliable method to experimentally detect and assess the acrosome reaction in mouse spermatozoa. PNA-FITC labelling was restricted to the acrosome and was not influenced by the fixation procedure; either absolute methanol or paraformaldehyde. In contrast, PSA-FITC not only labelled the acrosome, but also the whole head and the flagellum. This aspect was especially marked after methanol fixation. The cytoplasmic droplet, when present, was also stained by PSA-FITC. Incubation with the calcium ionophore ionomycin induced a concentration and time-dependent increase in the number of acrosome reactions. Compared to spotted preparations, smear samples exhibited a high proportion of spermatozoa with damaged acrosome. In conclusion, PNA-FITC labelling was more accurate than PSA-FITC labelling to detect the acrosome of mouse spermatozoa. The fixation method (methanol vs. paraformaldehyde) had no influence on the staining pattern of PNA-FITC labelling, but spotted preparations are recommended to avoid mechanical damage to the acrosome. Ionophore challenge confirmed the existence of a calcium-dependent acrosome reaction in mouse spermatozoa and validated the use of PNA-FITC to quantify this physiological process. The present study illustrates important methodological considerations which need to be taken into account in order to design a reliable and reproducible protocol for the study of the acrosome reaction.


Subject(s)
Acrosome Reaction , Acrosome/metabolism , Peanut Agglutinin/metabolism , Spermatozoa/cytology , Spermatozoa/metabolism , Acrosome/drug effects , Animals , Dose-Response Relationship, Drug , Fluorescein-5-isothiocyanate/metabolism , Fluorescent Dyes/metabolism , Ionomycin/pharmacology , Ionophores/pharmacology , Male , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Reproducibility of Results , Staining and Labeling/methods
2.
Int J Mol Med ; 18(6): 1047-55, 2006 Dec.
Article in English | MEDLINE | ID: mdl-17089007

ABSTRACT

Second generation rats depleted in long-chain polyunsaturated omega3 fatty acids are currently used as an animal model for the insufficient dietary supply of such fatty acids often prevailing in Western populations. The present study deals mainly with the effects of a novel medium-chain triglyceride: fish oil emulsion (MCT:FO), as compared to a control medium-chain triglyceride:olive oil emulsion (MCT: OO), administered as an intravenous bolus to the omega3-depleted rats 60-120 min before sacrifice upon selected biochemical and biophysical variables. The major findings consisted of a severe decrease of the omega3 fatty acid content of liver lipids in non-injected omega3-depleted rats and its partial correction after injection of the MCT:FO emulsion. The omega3-depleted rats also displayed liver steatosis, increased incorporation of long-chain polyunsaturated omega6 fatty acids in liver phospholipids and increased activity of liver Delta9-desaturase. As judged from the effects of ouabain upon 86Rb net uptake by isolated pancreatic islets, the activity of Na+,K+-ATPase was virtually abolished in the omega3-depleted rats. The latter defect was corrected by prior intravenous injection of the MCT:FO emulsion, this coinciding with suppression of the excessive secretory response to a number of insulin secretagogues otherwise observed in the islets of omega3-depleted rats injected or not with the MCT:OO emulsion.


Subject(s)
Cations/metabolism , Fat Emulsions, Intravenous/pharmacology , Fatty Acids, Omega-3/physiology , Islets of Langerhans/physiology , Triglycerides/chemistry , Triglycerides/pharmacology , Animals , Fat Emulsions, Intravenous/administration & dosage , Fat Emulsions, Intravenous/chemistry , Fatty Acids, Nonesterified/blood , Fatty Acids, Nonesterified/chemistry , Fatty Acids, Omega-3/analysis , Fatty Acids, Omega-3/metabolism , Fatty Acids, Unsaturated/biosynthesis , Fatty Acids, Unsaturated/chemistry , Female , Fish Oils/chemistry , Islets of Langerhans/metabolism , Liver/chemistry , Liver/metabolism , Rats , Triglycerides/administration & dosage , Triglycerides/blood
3.
Ann Otol Rhinol Laryngol ; 111(12 Pt 1): 1097-107, 2002 Dec.
Article in English | MEDLINE | ID: mdl-12498371

ABSTRACT

We characterized the anti-inflammatory effects of budesonide on the expression of adhesion molecules involving Lewis(a) (Le(a)) epitope, its sialylated derivative (sLe(a)), and their respective binding sites in human nasal polyposis. By computer-assisted microscopy, we quantitatively characterized the level of histochemical expression of L- and P-selectins, sialylated and nonsialylated Le(a) epitopes, and their respective binding sites in both surface epithelium and glandular epithelium of human nasal polyps obtained from surgical resection, maintained under ex vivo tissue culture conditions for 24 hours, and treated or not with budesonide. Intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) were chosen as methodological controls, because data already published in the literature clearly indicated budesonide-mediated effects on ICAM-1 and VCAM-1 levels of expression. The present data show that budesonide significantly modified the levels of expression of ICAM-1 and VCAM-1, and to a lesser extent that of P-selectin, in the surface and glandular epithelia. Budesonide markedly decreased the levels of expression of the binding sites for both Le(a) and sLe(a), while those of Le(a) and sLe(a) remained globally unchanged. In conclusion, the present study documents that glucocorticoid-induced effects can encompass receptors for Le(a) epitopes different from E- and P-selectins on epithelial cells of human nasal polyps.


Subject(s)
Anti-Inflammatory Agents/immunology , Budesonide/immunology , E-Selectin/drug effects , Gangliosides/immunology , Gene Expression Regulation/drug effects , Gene Expression Regulation/immunology , Lewis Blood Group Antigens/immunology , Nasal Polyps/drug therapy , Nasal Polyps/immunology , P-Selectin/drug effects , Anti-Inflammatory Agents/pharmacology , Binding Sites, Antibody/drug effects , Budesonide/pharmacology , CA-19-9 Antigen , Culture Techniques , Drug Evaluation, Preclinical , E-Selectin/analysis , Eosinophils/immunology , Epitopes , Gangliosides/analysis , Humans , Hypersensitivity/complications , Hypersensitivity/diagnosis , Immunohistochemistry , Intercellular Adhesion Molecule-1/analysis , Intercellular Adhesion Molecule-1/drug effects , Lewis Blood Group Antigens/analysis , Nasal Polyps/etiology , Nasal Polyps/pathology , P-Selectin/analysis , Vascular Cell Adhesion Molecule-1/analysis , Vascular Cell Adhesion Molecule-1/drug effects
4.
Biochim Biophys Acta ; 1572(2-3): 285-93, 2002 Sep 19.
Article in English | MEDLINE | ID: mdl-12223276

ABSTRACT

The galectins are a family of proteins that are distributed widely in all living organisms. All of them share galactose-specificity. At present, 14 members of the family are characterized in mammals. The galectins have been implicated in many essential functions including development, differentiation, cell-cell adhesion, cell-matrix interaction, growth regulation, apoptosis, RNA splicing, and tumor metastasis. Although efforts have mostly focused on the possible function of galectins in tumor development and invasiveness, their precise role in this field is still debated. This review discusses the recent way in which the expression of galectins and galectin-binding sites may affect the behavior of a variety of human neoplastic tissues.


Subject(s)
Carcinoma/etiology , Hemagglutinins/physiology , Neoplasms/etiology , Adipose Tissue/metabolism , Animals , Biomarkers, Tumor/analysis , Carcinoma/diagnosis , Carcinoma/metabolism , Galectins , Hemagglutinins/biosynthesis , Hemagglutinins/metabolism , Humans , Lectins/analysis , Lymphoid Tissue/metabolism , Muscles/metabolism , Neoplasms/diagnosis , Neoplasms/metabolism , Prognosis , Tumor Cells, Cultured
5.
J Neuropathol Exp Neurol ; 61(7): 585-96, 2002 Jul.
Article in English | MEDLINE | ID: mdl-12125737

ABSTRACT

We show that high-grade astrocytic tumors with high levels of galectin-1 expression are associated with dismal prognoses. The immunohistochemical analysis of galectin-1 expression of human U87 and U373 glioblastoma xenografts from the brains of nude mice revealed a higher level of galectin-1 expression in invasive areas rather than non-invasive areas of the xenografts. Nude mice intracranially grafted with U87 or U373 cells constitutively expressing low levels of galectin-1 (by stable transfection of an expression vector containing the antisense mRNA of galectin-1) had longer survival periods than those grafted with U87 or U373 cells expressing normal levels of galectin-1. Galectin-1 added to the culture media markedly and specifically increased cell motility levels in human neoplastic astrocytes. These effects are related to marked modifications in the organization of the actin cytoskeleton and the increase in small GTPase RhoA expression. All the data obtained indicate that galectin-1 enhances the migratory capabilities of tumor astrocytes and, therefore, their biological aggressiveness.


Subject(s)
Actin Cytoskeleton/metabolism , Brain Neoplasms/metabolism , Cell Movement/physiology , Glioblastoma/metabolism , Hemagglutinins/metabolism , Neoplasm Invasiveness/physiopathology , Tumor Cells, Cultured/metabolism , rhoA GTP-Binding Protein/metabolism , Actin Cytoskeleton/drug effects , Actin Cytoskeleton/pathology , Animals , Brain/metabolism , Brain/pathology , Brain/surgery , Brain Neoplasms/pathology , Brain Neoplasms/physiopathology , Brain Tissue Transplantation , Cell Movement/drug effects , Disease Progression , Female , Galectin 1 , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/physiology , Glioblastoma/pathology , Glioblastoma/physiopathology , Graft Survival/drug effects , Graft Survival/physiology , Hemagglutinins/genetics , Hemagglutinins/pharmacology , Humans , Male , Mice , Mice, Nude , Neoplasm Invasiveness/pathology , RNA, Antisense/genetics , Survival Rate , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/transplantation , rhoA GTP-Binding Protein/drug effects
6.
Lab Invest ; 82(2): 147-58, 2002 Feb.
Article in English | MEDLINE | ID: mdl-11850528

ABSTRACT

Because of the importance of galectins for various cellular activities, the influence of the glucocorticoid budesonide on the level of expression of galectins-1 and -3 was investigated in human nasal polyposis. Ten nasal polyps obtained from surgical resection were maintained for 24 hours in the presence of various concentrations of budesonide. As quantitatively demonstrated by means of computer-assisted microscopy, 250 ng/ml (the highest dose tested) induced a pronounced increase of galectin-1 expression. This feature was observed in nasal polyps from allergic patients but not in those from nonallergic patients. Since eosinophils represent the main inflammatory cell population in nasal polyps, we investigated the effect of galectin-1 on their migration levels by means of quantitative phase-contrast computer-assisted videomicroscopy. Our results show that galectin-1 (coated on plastic supports) markedly reduced the migration levels of eosinophils in comparison to P-selectin. On the cellular level, marked modifications in the polymerization/depolymerization dynamics of the actin cytoskeleton (as revealed by means of computer-assisted fluorescence microscopy) and, to a much lesser extent, an increase in the adhesiveness of eosinophils to tested substrata were detectable. The present study therefore reveals a new galectin-1-mediated mechanism of action for glucocorticoid-mediated anti-inflammatory effects.


Subject(s)
Anti-Inflammatory Agents/pharmacology , Budesonide/pharmacology , Cell Movement/physiology , Eosinophils/cytology , Hemagglutinins/metabolism , Nasal Polyps/metabolism , Administration, Topical , Antigens, Differentiation/metabolism , Binding Sites , Biopolymers , Blotting, Western , Cell Adhesion/physiology , Culture Techniques , Galectin 1 , Galectin 3 , Glucocorticoids , Hemagglutinins/physiology , Humans , Reverse Transcriptase Polymerase Chain Reaction
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