Heparin and Heparan Sulfate: Analyzing Structure and Microheterogeneity [chapter]

available in PMC 2013 August 28The structural microheterogeneity of heparin and heparan sulfate is one of the major reasons for the multifunctionality exhibited by this class of molecules. In a physiological context, these molecules primarily exert their effects extracellularly by mediating key processes of cellular cross-talk and signaling leading to the modulation of a number of different biological activities including development, cell proliferation, and inflammation. This structural diversity is biosynthetically imprinted in a nontemplate-driven manner and may also be dynamically remodeled as cellular function changes. Understanding the structural information encoded in these molecules forms the basis for attempting to understand the complex biology they mediate. This chapter provides an overview of the origin of the structural microheterogeneity observed in heparin and heparan sulfate, and the orthogonal analytical methodologies that are required to help decipher this information

Analysis and characterization of heparin impurities

This review discusses recent developments in analytical methods available for the sensitive separation, detection and structural characterization of heparin contaminants. The adulteration of raw heparin with oversulfated chondroitin sulfate (OSCS) in 2007–2008 spawned a global crisis resulting in extensive revisions to the pharmacopeia monographs on heparin and prompting the FDA to recommend the development of additional physicochemical methods for the analysis of heparin purity. The analytical chemistry community quickly responded to this challenge, developing a wide variety of innovative approaches, several of which are reported in this special issue. This review provides an overview of methods of heparin isolation and digestion, discusses known heparin contaminants, including OSCS, and summarizes recent publications on heparin impurity analysis using sensors, near-IR, Raman, and NMR spectroscopy, as well as electrophoretic and chromatographic separations

Molecular size-dependent specificity of hyaluronan on functional properties, morphology and matrix composition of mammary cancer cells.

High levels of hyaluronan (\u397\u391), a major extracellular matrix (ECM) glycosaminoglycan, have been correlated with poor clinical outcome in several malignancies, including breast cancer. The high and low molecular weight H\u391 forms exert diverse biological functions. Depending on their molecular size, \u397\u391 forms either promote or attenuate signaling cascades that regulate cancer progression. In order to evaluate the effects of different \u397\u391 forms on breast cancer cells' behavior, \u397\u391 fragments of defined molecular size were synthesized. Breast cancer cells of different estrogen receptor (ER) status \u2013 the low metastatic, ER\u3b1-positive MCF-7 epithelial cells and the highly aggressive, ER\u3b2-positive MDA-MB-231 mesenchymal cells \u2013 were evaluated following treatment with HA fragments. Scanning electron microscopy revealed that HA fragments critically affect the morphology of breast cancer cells in a molecular-size dependent mode. Moreover, the \u397\u391 fragments affect cell functional properties, the expression of major ECM mediators and epithelial-to-mesenchymal transition (\u395\u39c\u3a4) markers. Notably, treatment with 200\u202fkDa \u397\u391 increased the expression levels of the epithelial marker \u395-cadherin and reduced the expression levels of HA synthase 2 and mesenchymal markers, like fibronectin and snail2/slug. These novel data suggest that the effects of HA in breast cancer cells depend on the molecular size and the ER status. An in-depth understanding on the mechanistic basis of these effects may contribute on the development of novel therapeutic strategies for the pharmacological targeting of aggressive breast cancer

Diagnostic value of bone remodeling markers in the diagnosis of bone metastases in patients with breast cancer

Metastatic spread to bone is common in patients with breast cancer and its early detection is required for the better management of these patients. Several biochemical markers of bone remodeling have been recently developed, in order to assess metastatic bone disease with non radiologic methods. The pyridinolin cross-linked amino-terminal telopeptide of type I collagen (NTx) has been measured in serum and urine as a specific marker of bone collagen breakdown, while the bone-isoform of alkaline phosphatase (BAP) has been used to determine bone formation activity. Thirty-three consecutive ambulatory patients with metastatic breast cancer and bone metastases and 31 with extraskeletal metastases only, matched for age and menopausal status, were studied. Serum levels of NTx and BAP were measured by enzyme-linked immunosorbent assays. The diagnostic accuracy of both markers was evaluated by receiver operating characteristic (ROC) analysis. Patients with bone metastases had significantly higher levels of NTx (37.0 +/- 36.9 nM BCE versus 23.5 +/- 21.0 nM BCE, P < 0.05) and BAP (57.8 +/- 31.7 U/L versus 36.5 +/- 28.5 U/L, P < 0.01) compared to those without bone metastases. NTx was positively correlated with BAP (R = 0.340, P < 0.01). The area under the ROC curve was 0.671 for NTx and 0.755 for BAP. Using a cut-off value of 29.7 nM BCE for NTx, specificity and sensitivity were 87.1% and 45.5%, respectively; in the case of BAP, using a cut-off value of 50.6 U/L, the specificity and sensitivity were 90.3% and 54.5%, respectively. In patients not receiving concomitant hormonal treatment, the area under the ROC curve was 0.724 for NTx and 0.822 for BAP; in this subgroup of patients, using a cut-off value of 30.0 nM BCE for NTx, the specificity and sensitivity were 96.2% and 47.1%, respectively, while using a cut-off value of 50.0 U/L for BAP, the corresponding percentages were 92.3% and 70.6%. 1 Although serum NTx and BAP are quite specific, they are not sensitive enough to diagnose bone metastases in patients with advanced breast cancer. Their diagnostic accuracy, however, is considerably enhanced in patients not receiving hormonal therapy. (C) 2004 Elsevier B.V. All rights reserved

Proteoglycan chemical diversity drives multifunctional cell regulation and therapeutics

The extracellular matrix (ECM) constitutes a highly dynamic three-dimensional structural network comprised of macromolecules, such as proteoglycans/glycosaminoglycans (PGs/GAGs), collagens, laminins, fibronectin, elastin, other glycoproteins and proteinases. In recent years, the field of PGs has expanded rapidly. Due to their high structural complexity and heterogeneity, PGs mediate several homeostatic and pathological processes. PGs consist of a protein core and one or more covalently attached GAG chains, which provide the protein cores with the ability to interact with several proteins. The GAG building blocks of PGs significantly influence the chemical and functional properties of PGs. The primary goal of this comprehensive review is to summarize major achievements and paradigm-shifting discoveries made on the PG/GAG chemistry-biology axis, focusing on structural variability, structure-function relationships, metabolic, molecular, and epigenetic mechanisms underlying their synthesis. Recent insights related to exosome biogenesis, degradation, and cell signaling, their status as diagnostic tools and potential pharmacological targets in diseases as well as current applications in nanotechnology and biotechnology are addressed. Moreover, issues related to docking studies, molecular modeling, GAG/PG interaction networks, and their integration are discussed

Proteoglycan Chemical Diversity Drives Multifunctional Cell Regulation and Therapeutics.

The extracellular matrix (ECM) constitutes a highly dynamic three-dimensional structural network comprised of macromolecules, such as proteoglycans/glycosaminoglycans (PGs/GAGs), collagens, laminins, fibronectin, elastin, other glycoproteins and proteinases. In recent years, the field of PGs has expanded rapidly. Due to their high structural complexity and heterogeneity, PGs mediate several homeostatic and pathological processes. PGs consist of a protein core and one or more covalently attached GAG chains, which provide the protein cores with the ability to interact with several proteins. The GAG building blocks of PGs significantly influence the chemical and functional properties of PGs. The primary goal of this comprehensive review is to summarize major achievements and paradigm-shifting discoveries made on the PG/GAG chemistry-biology axis, focusing on structural variability, structure\u2013function relationships, metabolic, molecular, and epigenetic mechanisms underlying their synthesis. Recent insights related to exosome biogenesis, degradation, and cell signaling, their status as diagnostic tools and potential pharmacological targets in diseases as well as current applications in nanotechnology and biotechnology are addressed. Moreover, issues related to docking studies, molecular modeling, GAG/PG interaction networks, and their integration are discussed

Capillary electrophoresis applied to polysaccharide characterization

Capillary electrophoresis is a consolidated analytical approach for the structural characterization of polysaccharide mono- and oligomer constituents, as demonstrated in this chapter, which surveys several applications of this technique on chemically and enzymatically degraded polysaccharides, covering the last 10 to 12 years Capillary electrophoresis is also demonstrated to be highly reliable for determination of polysaccharides in biological samples, as it analyzes quite complex matrices even without any pretreatment, a distinctive feature with respect to other separative strategies The versatility of this technique is clearly demonstrated by its potential in evaluating macromolecular features of polysaccharides, such as size (molecular weight, chain length), chain rigidity, charge density, and chemical modification

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 I at the University of Athens; (d) oxidative stress-induced DNA damage and the role of oncogenes in senescence at the University of Ioannina; (c) studies in the connective tissue at the University of Patras; (f) protcomic studies at the Biomedical Sciences Research Center ‘Alexander Fleming’; (g) work on Caenorhabditis clegans at the Foundation for Research and Technolog;(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. These research activities are well recognized by the international scientific community as it is evident by the group’s very good publication records as well as by their direct funding from both European Union and USA. This article summarizes these research activities and discuss future directions and efforts towards the further development of the ageing field in Greece. (C) 2002 Elsevier Science Inc. All rights reserved