Amanda Weyers

Polysaccharides and phytochemicals: a natural reservoir for the green synthesis of gold and silver nanoparticles

Currently, sustainability initiatives that use green chemistry to improve and/or protect our global environment are becoming focal issues in many fields of research. Instead of using toxic chemicals for the reduction and stabilisation of metallic nanoparticles, the use of various biological entities has received considerable attention in the field of nanobiotechnology. Among the many possible natural products, polysaccharides and biologically active plant products represent excellent scaffolds for this purpose. Polysaccharides have hydroxyl groups, a hemiacetal reducing end, and other functionalities that can play important roles in both the reduction and the stabilisation of metallic nanoparticles. Among the various categories of compounds in plants that have potent biological activities, phytochemicals are emerging as an important natural resource for the synthesis of metallic nanoparticles. The focus of this review is the application of polysaccharides and phytochemicals in the green synthesis of gold and silver nanoparticles to afford biocomposites with novel uses in nanomedicine and as nanocomposites.

Disaccharide analysis of glycosaminoglycan mixtures by ultra-high-performance liquid chromatography–mass spectrometry

Glycosaminoglycans are a family of polysaccharides widely distributed in all eukaryotic cells. These polyanionic, linear chain polysaccharides are composed of repeating disaccharide units that are often differentially substituted with sulfo groups. The diversity of glycosaminoglycan structures in cells, tissues and among different organisms reflect their functional an evolutionary importance. Glycosaminoglycan composition and structure also changes in development, aging and in disease progression, making their accurate and reliable analysis a critical, albeit, challenging endeavor. Quantitative disaccharide compositional analysis is one of the primary ways to characterize glycosaminoglycan composition and structure and has a direct relationship with glycosaminoglycan biological functions. In this study, glycosaminoglycan disaccharides, prepared from heparan sulfate/heparin, chondroitin sulfate/dermatan sulfate and neutral hyaluronic acid using multiple polysaccharide lyases, were fluorescently labeled with 2-aminoacridone, fractionated into 17 well-resolved components by reverse-phase ultra-performance liquid chromatography, and analyzed by electrospray ionization mass spectrometry. This analysis was successfully applied to cell, tissue, and biological fluid samples for the picomole level detection of glycosaminoglycan composition and structure.

Ultra-performance ion-pairing liquid chromatography with on-line electrospray ion trap mass spectrometry for heparin disaccharide analysis

A high-resolution method for the separation and analysis of disaccharides prepared from heparin and heparan sulfate (HS) using heparin lyases is described. Ultra-performance liquid chromatography in a reverse-phase ion-pairing mode efficiently separates eight heparin/HS disaccharides. The disaccharides can then be detected and quantified using electrospray ionization mass spectrometry. This method is particularly useful in the analysis of small amounts of biological samples, including cells, tissues, and biological fluids, because it provides high sensitivity without being subject to interference from proteins, peptides, and other sample impurities.

Isolation of bovine corneal keratan sulfate and its growth factor and morphogen binding

Keratan sulfate (KS) is an important glycosaminoglycan that is found in cartilage, reproductive tissues, and neural tissues. Corneal KS glycosaminoglycan is found N‐linked to lumican, keratocan and mimecan proteoglycans, and has been widely studied by investigators interested in corneal development and diseases. Recently, the availability of corneal KS has become severely limited, owing to restrictions on the shipment of bovine central nervous system byproducts across international borders in an effort to prevent additional cases of mad cow disease. We report a simple method for the purification of multi‐milligram quantities of bovine corneal KS, and characterize its structural properties. We also examined its protein‐binding properties, and discovered that corneal KS bound with high affinity to fibroblast growth factor‐2 and sonic hedgehog, a growth factor and a morphogen involved in corneal development and healing.

Neoproteoglycans in tissue engineering

Proteoglycans, comprised of a core protein to which glycosaminoglycan chains are covalently linked, are an important structural and functional family of macromolecules found in the extracellular matrix. Advances in our understanding of biological interactions have lead to a greater appreciation for the need to design tissue engineering scaffolds that incorporate mimetics of key extracellular matrix components. A variety of synthetic and semisynthetic molecules and polymers have been examined by tissue engineers that serve as structural, chemical and biological replacements for proteoglycans. These proteoglycan mimetics have been referred to as neoproteoglycans and serve as functional and therapeutic replacements for natural proteoglycans that are often unavailable for tissue engineering studies. Although neoproteoglycans have important limitations, such as limited signaling ability and biocompatibility, they have shown promise in replacing the natural activity of proteoglycans through cell and protein binding interactions. This review focuses on the recent in vivo and in vitro tissue engineering applications of three basic types of neoproteoglycan structures, protein–glycosaminoglycan conjugates, nano‐glycosaminoglycan composites and polymer–glycosaminoglycan complexes.

Impact of autoclave sterilization on the activity and structure of formulated heparin

The stability of a formulated heparin was examined during its sterilization by autoclaving. A new method to follow loss in heparin binding to the serine protease inhibitor, antithrombin III, and the serine protease, thrombin, was developed using a surface plasmon resonance competitive binding assay. This loss in binding affinity correlated well with loss in antifactor IIa (thrombin) activity as well as antifactor Xa activity as measured using conventional amidolytic assays. Autoclaving also resulted in a modest breakdown of the heparin backbone as confirmed by a slight reduction in number-averaged and weight-averaged molecular weight and an increase in polydispersity. Although no clear changes were observed by nuclear magnetic resonance spectroscopy, disaccharide composition analysis using high-performance liquid chromatography-electrospray ionization-mass spectrometry suggested that loss of selected sulfo groups had taken place. It is this sulfo group loss that probably accounts for a decrease in the binding of autoclaved heparin to antithrombin III and thrombin as well as the observed decrease in its amidolytic activity.

Carbohydrate-Containing Molecules as Potential Biomarkers in Colon Cancer

Glycans play a critical role in physiological and pathological processes through interaction with a variety of ligands. Altered expression and dysregulation of these molecules can cause aberrant cellular function such as malignancy. Glycomics provide information of the structure and function of glycans, glycolipids, and glycoproteins such as proteoglycans, and may help to predict cancer development and progression as biomarkers. In this report, we compared the expression of proteoglycans, the content and structure of glycosaminoglycans and glycolipids between patient-matched normal and cancer tissues obtained from colon cancer patients. Tumor-related proteoglycans, glypican-3, and syndecan-1 showed downregulation in cancer tissues compared to normal tissues. In cancer tissue, the total amount of chondroitin sulfate (CS)/dermatan sulfate and heparan sulfate were lower and, interestingly, the level of disaccharide units of both 4S6S (CS-E) and 6S (CS-C) were higher compared to normal tissue. Also, overall lipids including glycolipids, a major glycomics target, were analyzed by hydrophilic interaction liquid chromatography mass spectrometry. Increase of lyso-phosphatidylcholine (phospholipid), sphingomyelin (sphigolipid), and four types of glycolipids (glucosylceramide, lactosylceramide, monosialic acid ganglioside, and globoside 4) in cancer tissue showed the possibility as potential biomarkers in colon cancer. While requiring the need for careful interpretation, this type of broad investigation gives us a better understanding of pathophysiological roles on glycosaminoglycans and glycolipids and might be a powerful tool for colon cancer diagnosis.

Ligand Environment of the S2 State of Photosystem II: A Study of the Hyperfine Interactions of the Tetranuclear Manganese Cluster by 2D 14N HYSCORE Spectroscopy

The solar water-splitting protein complex, photosystem II, catalyzes the light-driven oxidation of water to dioxygen in Nature. The four-electron oxidation reaction of water occurs at the tetranuclear manganese-calcium-oxo catalytic cluster that is present in the oxygen-evolving complex of photosystem II. The mechanism of light-driven water oxidation has been a subject of intense interest, and the oxygen-evolving complex of photosystem II has been studied extensively by structural and biochemical methods. While the recent X-ray crystal structures and single-crystal EXAFS investigations provide a model for the geometry of the tetranuclear manganese-calcium-oxo catalytic cluster, there is limited knowledge of the protein environment that surrounds the catalytic cluster. In this study, we demonstrate the application of two-dimensional hyperfine sublevel correlation spectroscopy to determine the magnetic couplings of the catalytic cluster with the (14)N atoms of surrounding amino acid residues in the S(2) state of the oxygen-evolving complex of photosystem II. We utilize two-dimensional difference spectroscopy to facilitate unambiguous assignments of the spectral features and identify at least three separate (14)N atoms that are interacting with the catalytic cluster in the S(2) state. The results presented here, for the first time, identify previously unknown ligands to the catalytic cluster of photosystem II and provide avenues for the assignment of residues by site-directed mutagenesis and the refinement of computational and mechanistic models of photosystem II.

New Functional Tools for Antithrombogenic Activity Assessment of Live Surface Glycocalyx

Objective—It is widely accepted that the presence of a glycosaminoglycan-rich glycocalyx is essential for endothelialized vasculature health; in fact, a damaged or impaired glycocalyx has been demonstrated in many vascular diseases. Currently, there are no methods that characterize glycocalyx functionality, thus limiting investigators’ ability to assess the role of the glycocalyx in vascular health. Approach and Results—We have developed novel, easy-to-use, in vitro assays that directly quantify live endothelialized surface’s functional heparin weights and their anticoagulant capacity to inactivate Factor Xa and thrombin. Using our assays, we characterized 2 commonly used vascular models: native rat aorta and cultured human umbilical vein endothelial cell monolayer. We determined heparin contents to be ≈10 000 ng/cm2 on the native aorta and ≈10-fold lower on cultured human umbilical vein endothelial cells. Interestingly, human umbilical vein endothelial cells demonstrated a 5-fold lower anticoagulation capacity in inactivating both Factor Xa and thrombin relative to native aortas. We verified the validity and accuracy of the novel assays developed in this work using liquid chromatography–mass spectrometry analysis. Conclusions—Our assays are of high relevance in the vascular community because they can be used to establish the antithrombogenic capacity of many different types of surfaces such as vascular grafts and transplants. This work will also advance the capacity for glycocalyx-targeting therapeutics development to treat damaged vasculatures.

Capillary Electrophoresis for the Analysis of Glycosaminoglycan-Derived Disaccharides

Capillary electrophoresis is a common technique used for glycosaminoglycan-derived disaccharide analysis because of its high resolving power, high separation efficiency, high sensitivity, short analysis time, and straightforward operation. CE coupled to laser-induced fluorescence (LIF) detection shows an approximately 100 times higher sensitivity than traditional UV detection at 232 nm. 2-Aminoacridone (AMAC) is a widely used fluorophore for labeling unsaturated disaccharides by deductive amination, which is one of the most important method of derivatization of disaccharides for CE-LIF detection. Outlined in this chapter is a protocol of analyzing glycosaminoglycan-derived disaccharides by CE-LIF with AMAC derivatization.