Separation methods for sialic acids and critical evaluation of their biologic relevance.

Sialic acids are biosynthesized by almost all organisms as a 9-carbon carboxylated monosaccharide and are integral components of glycoconjugates. More than 40 naturally occurring sialic acid derivatives of the three main forms of sialic acids, the N-acetyl- and N-glycolylneuraminic acid and 2-keto-3-deoxy-nonulosonic acid have been identified. Due to the great importance of sialic acids as key mediators in a plethora of cellular events, including cell-cell recognition and cell-matrix interactions, their analysis in biologic samples is useful for a deeper understanding of the various (patho)physiological processes and of value in disease diagnosis and monitoring.

Decreased biglycan expression and differential decorin localization in human abdominal aortic aneurysms.

The hallmark feature of abdominal aortic aneurysm (AAA) is the progressive degeneration of aortic wall. Matrix proteoglycans (PGs) play important roles in the development of vascular diseases and the function of the tissue. In this study, we examined the concentration, expression and localization of the small extracellular matrix PG biglycan and decorin.

Ageing research in Greece.

Ageing research in Greece is well established. Research groups located in universities, research institutes or public hospitals are studying various and complementary aspects of ageing. These research activities include (a) functional analysis of Clusterin/Apolipoprotein J, studies in healthy centenarians and work on protein degradation and the role of proteasome during senescence at the National Hellenic Research Foundation; (b) regulation of cell proliferation and tissue formation, a nationwide study of determinants and markers of successful ageing in Greek centenarians and studies of histone gene expression and acetylation at the National Center for Scientific Research, Demokritos; (c) work on amyloid precursor protein and Presenilin 1 at the University of Athens; (d) oxidative stress-induced DNA damage and the role of oncogenes in senescence at the University of Ioannina; (e) studies in the connective tissue at the University of Patras; (f) proteomic studies at the Biomedical Sciences Research Center Alexander Fleming; (g) work on Caenorhabditis elegans at the Foundation for Research and Technology; (h) the role of ultraviolet radiation in skin ageing at Andreas Sygros Hospital; (i) follow-up studies in healthy elderly at the Athens Home for the Aged; and (j) socio-cultural aspects of ageing at the National School of Public Health.

Pig vitreous gel: macromolecular composition with particular reference to hyaluronan-binding proteoglycans.

The aim of this study was to examine the macromolecular composition of pig vitreous body with particular emphasis on hyaluronan-binding proteoglycans. The whole pig vitreous gel was found to contain 76 microg of hyaluronan-derived uronic acid, 700 microg of total protein and 150 microg of collagen per ml of gel. The contents of neutral hexoses and sialic acids were 80 and 22 microg/ml of vitreous gel, but only a minor proportion of them were found to be associated with the proteoglycan fraction.

Levels of specific antibodies towards the major antigenic determinant of slime-producing Staphylococcus epidermidis determined by an enzyme immunoassay and their protective effect in experimental keratitis.

Staphylococcus epidermidis is an important cause of bacterial keratitis. Certain S. epidermidis strains produce an extracellular slime layer rich in an acidic polysaccharide with a molecular size of 20 kDa (20-kDa PS).

Determination of twelve heparin- and heparan sulfate-derived disaccharides as 2-aminoacridone derivatives by capillary zone electrophoresis using ultraviolet and laser-induced fluorescence detection.

In quest for high sensitivities, we developed an ultrahigh capillary electrophoresis (CE) method for the structural analysis of heparin and heparan sulfate (HS) in biologic samples. Heparin and HS were digested with an equi-unit mixture of heparin lyases I, II and III and the obtained Delta-disaccharides were derivatized with the fluorophore 2-aminoacridone. All known twelve non-, mono-, di- and trisulfated Delta-disaccharides were completely resolved in a single run, using 50 mM phosphate buffer, pH 3.

Identification of kappa and lambda chains of the major immunoglobulin G subclasses by capillary zone electrophoresis.

Immunoglobulins are present in most tissues and plasma and play crucial role in immune system. Alteration of the levels of the immunoglobulin G (IgG) subclasses (IgG1, IgG2, IgG3 and IgG4) is an indication of a disturbed immunological response. The aim of the present study was the development of a capillary electrophoresis (CE) method for the analysis of IgG subclasses in respect to their variable kappa and lambda chains.

A capillary zone electrophoresis method for determining N-acetylneuraminic acid in glycoproteins and blood sera.

A simple capillary zone electrophoresis (CZE) method for the determination of the content of the major sialic acid form N-acetylneuraminic acid (Neu5Ac) in glycoproteins was established. The present method utilizes a simplified hydrolysis-purification procedure consisting of mild acid hydrolysis (25 mM trifluoroacetic acid for 2h at 80 degrees C) to release Neu5Ac and ultrafiltration on Centricon-3 membrane to remove the obtained asialoglycoproteins and other macromolecules present in biologic samples. Derivatization with benzoic anhydride at 80 degrees C for 20 min resulted in complete conversion of Neu5Ac to per-O-benzoylated Neu5Ac.

Structure analysis of lipoglycans and lipoglycan-derived carbohydrates by capillary electrophoresis and mass spectrometry.

Lipoglycans (lipopolysaccharides, lipoarabinomannans and glycolipids) are unique components of the cell membrane of all cells and the envelope of many bacteria. They play important roles in determining cell-environment interactions, which, however, are only partly understood due to incomplete description of their structural components, lipids and glycans. Capillary electrophoresis is an analytical technique of high separation efficiency and minimum sample requirements and has successfully been used for the analysis of several molecules of biological importance: proteins, nucleic acids and glycoconjugates.

Analysis of glycosaminoglycan-derived disaccharides in biologic samples by capillary electrophoresis and protocol for sequencing glycosaminoglycans.

Glycosaminoglycans are biologically significant carbohydrates which either as free chains (hyaluronan) or constituents of proteoglycans (chondroitin/dermatan sulfates, heparin, heparan sulfate and keratan sulfate) participate and regulate several cellular events and (patho)physiological processes. Capillary electrophoresis, due to its high resolving power and sensitivity, has been successfully used for the analysis of glycosaminoglycans. Determination of compositional characteristics, such as disaccharide sulfation pattern, is a useful prerequisite for elucidating the interactions of glycosaminoglycans with matrix effective molecules and, therefore, essential in understanding the biological functions of proteoglycans.

Increased aqueous humor basic fibroblast growth factor and hyaluronan levels in relation to the exfoliation syndrome and exfoliative glaucoma.

Purpose: To quantify the concentrations of basic fibroblast growth factor (bFGF) and hyaluronan (HA) in the aqueous humor of patients with the exfoliation syndrome (XFS) or exfoliative glaucoma (XFG).

Methods: Aqueous humor bFGF and HA levels were measured in 13 patients with XFS and in 7 patients with XFG. The results were compared with those obtained from 17 healthy controls.

Effects of mitogens on synthesis and distribution of heparan and chondroitin sulphates by human leukemic B, T cells and monocytes studied by high-performance liquid chromatography and radiochemical detection.

Human leukemic cell lines, Jurkat (T-cell leukemia), Daudi (Burkitt's lymphoma, B-cell leukemia) and THP-1 (acute monocytic leukemia) synthesize chondroitin sulphate (CS) and heparan sulphate (HS) in both cell membrane and culture medium. CS is the major secreted GAG in all cell lines, as well as the major cell-retarded glycosaminoglycan (GAG) in Jurkat and Daudi, whereas HS is the major GAG in the cell membrane of THP-1. The effects of mitogenic substances on both synthesis and distribution of GAGs in Jurkat, Daudi and THP-1, independently of their effect on cell proliferation, were studied.

Capillary electrophoresis: a superior miniaturized tool for analysis of the mono-, di-, and oligosaccharide constituents of glycan moieties in proteoglycans.

Proteoglycans are a major family of glycoconjugates which participate in and regulate several cellular events and biological functions. Their glycan chains determine their physicochemical and biological properties. Capillary electrophoresis, because of its high resolving power and sensitivity, has been successfully used for the analysis of carbohydrates.

Synthesis and distribution of glycosaminoglycans in human leukemic B- and T-cells and monocytes studied using specific enzymic treatments and high-performance liquid chromatography.

Identification of glycosaminoglycans (GAGs) synthesized by three human leukaemic cell lines-Jurkat (T-cell leukaemia), Daudi (Burkitt's lymphoma, B-cell leukaemia) and THP-1 (acute monocytic leukemia)-and normal peripheral blood mononuclear cells (PBMC) and their distribution among cell membrane and culture medium were studied. GAGs were isolated using ion-exchange chromatography on DEAE-Sephacel and their composition and fine chemical structure were studied using high-performance liquid chromatography with radiochemical detection. All cell lines synthesize chondroitin sulphate (CS) and heparan sulphate (HS) in both cell membrane and culture medium.

Microemulsion electrokinetic capillary chromatography of sulfated disaccharides derived from glycosaminoglycans.

Microemulsion electrokinetic capillary chromatography (MEEKC) is a capillary electrophoresis technique in which neutral and ionized species can be resolved according to their partitioning into moving oil droplets present in the operating buffer. In this report, we present for the first time the application of MEEKC in the analysis of glycosaminoglycans. An efficient method for the separation of the variously sulfated delta-disaccharides obtained following digestion of chondroitin and dermatan sulfates with chondro/ dermato lyases and derivatization with 2-aminoacridone is described.

Identification of oligomeric domains within dermatan sulfate chains using differential enzymic treatments, derivatization with 2-aminoacridone and capillary electrophoresis.

Galactosaminoglycans, i.e. dermatan sulfate (DS) and chondroitin sulfate, are linear heteropolysaccharides consisting of repeating disaccharide units of L-iduronic acid (L-IdoA) or D-glucuronic acid (D-GlcA) residues linked to N-acetyl-galactosamine.

Identification, quantification and fine structural characterization of glycosaminoglycans from uterine leiomyoma and normal myometrium.

The type, amount and fine chemical composition of glycosaminoglycans (GAGs) present both in human normal myometrium and uterine leiomyoma have been studied. GAGs were fractionated by ion-exchange chromatography on DEAE-Sephacel, isolated by gel-permeation chromatography on Sepharose CL-6B and characterized using electrophoresis in cellulose acetate membranes, specific enzymic treatments and analysis by high-performance capillary electrophoresis (HPCE). No statistical intrabatch differences in total GAG content in both tissues were identified, whereas significant interbatch differences between normal myometrium and uterine leiomyoma were recorded.

Determination of malondialdehyde by capillary electrophoresis, application to human plasma and relation of its levels with prematurity.

Malondialdehyde (MDA) is considered as the most important marker for monitoring lipid peroxidation, which is strongly associated with the development of serious diseases in adults and premature neonates. In this paper we report a method for determination of free MDA in human plasma using capillary zone electrophoresis. MDA was separated and determined as conjugate with tetrabutylammonium hydrogen sulphate (TBAS).

Application of capillary electrophoresis in the analysis of novel synthetic dideoxynucleoside analogues with potential anti-HIV activity.

The aim of this study was the development of a capillary electrophoretic method for the analysis of a series of novel synthetic dideoxynucleoside analogues with potential anti-HIV activity. These analogues consist of a tetrahydrofuranyl or a tetrahydropyranyl ring as the pseudosugar part and bear a hydroxyethyl side-chain and a nucleobase of the pyrimidine (eg thymine or uracil) or the purine (adenine) type with cis or trans configuration. Analysis of these derivatives was performed by capillary zone electrophoresis using 25 mM phosphate pH 3.

Immunoreactivity of 80-kDa peptidoglycan and teichoic acid-like substance of slime producing S. epidermidis and specificity of their antibodies studied by an enzyme immunoassay.

S. epidermidis is considered an important cause of nosocomial bacteraernia in immunocompromized hosts as well as the commonest agent of sepsis in patients with prosthetic devices. Pathogenesis is attributed to adherence and growth on bioniaterials facilitated by production of extracellular slime.

Human abdominal aortic aneurysm is characterized by decreased versican concentration and specific downregulation of versican isoform V(0).

Abdominal aortic aneurysm (AAA) is a common disease of human aorta with increased incidence. It is a complication to atherosclerosis and it is closely associated with alterations in extracellular macromolecules. In this study, the levels of mRNA for versican--the major extracellular arterial proteoglycan (PG)--present in AAA and normal aortas were evaluated by reverse-transcriptase polymerase chain reaction.

Immunochemical-based assays in the bioanalysis of immunoglobulins.

Intravenous immunoglobulin (IVIG) preparations consist of IgG derived from a pool of human plasma of healthy individuals and have been used as routine treatment of patients with primary and secondary immunodeficiencies, autoimmune, and/or inflammatory diseases. Emergence of new infectious agents and development of antibiotic resistance in many bacteria have posed serious problems in the treatment of infections. Since IVIGs contain natural antibodies that occur in the healthy population, their administration to immunocompromised hosts either as a prophylactic agent or as complementary treatment to the usual antimicrobial treatment have been studied.