Koen P. Vercruysse

Controlled chemical modification of hyaluronic acid: synthesis, applications, and biodegradation of hydrazide derivatives.

Controlled modification of the carboxylic acid moieties of hyaluronic acid with mono- and polyfunctional hydrazides leads to biochemical probes, biopolymers with altered physical and chemical properties, tethered drugs for controlled release, and crosslinked hydrogels as biocompatible scaffoldings for tissue engineering. Methods for polyhydrazide synthesis, for prodrug preparation, for hydrogel crosslinking, and for monitoring biodegradation are described.

Attachment of hyaluronic acid to polypropylene, polystyrene, and polytetrafluoroethylene

Surfaces of polypropylene (PP), polystyrene (PS) and polytetrafluoroethylene (PTFE) were activated with radio frequency plasmas Ar and NH3 to aminate the polymer surface and were subsequently reacted with hyaluronic acid (HA) in one of the three different attachment schemes. Results show that ammonia plasma treated polymers were more reactive toward HA attachment. The three chemistry schemes consisted of two distinct approaches: (1) direct attachment of the HA to the aminated surface, and (2) extending the reactive group away from the surface with succinic anhydride and then reacting the newly formed carboxylic acid group with an adipic dihydrazide modified HA (HA-ADH). The latter scheme proved to be more effective, suggesting that steric effects were involved with the reactivity of the HA with surface groups. These HA-coated polymers are a candidate for cell attachment and growth.

Control of enzymatic degradation of hyaluronan by divalent cations.

Enzymatic degradation of hyaluronan (HA) by testicular hyaluronidase (HAase, hyaluronate 4-glucanohydrolase) requires inclusion of mono- or divalent cations in the reaction mixture. Most divalent cations activated HAase with equal potency; however, Cu2+ suppressed degradation, and Ca2+ showed a concentration-dependent regulation of size of the oligosaccharide products. Careful selection of HAase assay parameters is critical for discovery of novel HAase inhibitors and for preparation of controlled-size oligosaccharide fragments.

Kinetic investigation of the degradation of hyaluronan by hyaluronidase using gel permeation chromatography.

A gel permeation chromatography (GPC) system for the analysis of hyaluronan was set up and calibrated by means of the universal calibration method. Using GPC, a kinetic assay of the action of hyaluronidase on hyaluronan has been developed. Applying Michaelis-Menten theory and the direct linear plot we have estimated the kinetic constants of the enzyme. By evaluating the decrease of the various molecular mass averages during the enzymatic reactions, we have given a demonstration of the at random degradation of hyaluronan by hyaluronidase in the initial stage of the reaction.

Interleukin-12 release from macrophages by hyaluronan, chondroitin sulfate A and chondroitin sulfate C oligosaccharides

Mixtures of hyaluronan (HA), chondroitin sulfate (CS)-A and CS-C oligosaccharides were generated through the enzymatic digestion of the polysaccharides with either mammalian hyaluronidase or bacterial HA lyase or chondroitinase. Compared to mammalian enzymes, bacterial enzymes hydrolyze the polysaccharides through a different mechanism yielding chemically distinct sets of oligosaccharides. Peripheral leukocytes and a human monocytic cell line were exposed to these oligosaccharides and the amount of interleukin-12 released by the cells was measured. For all types of oligosaccharide tested, we found that the amount of interleukin-12 induced by oligosaccharides generated with bacterial enzyme was significantly lower than the amount of interleukin-12 induced by oligosaccharides generated with mammalian enzyme. In addition, we observed that CS oligosaccharides generated with bacterial enzyme were capable of reducing the lipopolysaccharide-induced interleukin-12 production in macrophages. Our results indicate that HA or CS oligosaccharides generated with mammalian enzymes might possess pro-inflammatory potential, while HA or CS oligosaccharides generated with bacterial enzymes might possess non- or anti-inflammatory properties. The implications of our findings in view of the ongoing investigation of the potential therapeutic benefits of HA and CS in arthritis or other inflammatory pathologies are discussed.

Effects of Ascorbic Acid and Analogs on the Activity of Testicular Hyaluronidase and Hyaluronan Lyase on Hyaluronan

We have evaluated the inhibition of testicular hyaluronidase and hyaluronan lyase by L-ascorbic acid and chemical analogs. We observed that L-ascorbic acid, D-isoascorbic acid and dehydroascorbic acid inhibited both types of enzymes, but showed stronger effects towards hyaluronan lyase. But these compounds were observed to degrade the substrate, hyaluronan, by themselves. Of the other ascorbic acid analogs tested, saccharic acid inhibited hyaluronan lyase, while not affecting the enzymatic activity of testicular hyaluronidase, nor affecting the physic-chemical stability of hyaluronan. This is the first compound, to our knowledge, to be shown to possess such selective inhibition. Therefore, we propose that saccharic acid could serve as a lead compound for the development of potent and selective inhibitors of bacterial hyaluronan lyase or of polysaccharide lyase enzymes in general as we observed this compound to be capable of inhibiting chondroitinase ABC in addition to hyaluronan lyase.

Draft Genome Sequence of New Bacillus cereus Strain tsu1

ABSTRACT This paper reports the draft genome sequence of new Bacillus cereus strain tsu1, isolated on an agar-cellulose plate. The draft genome sequence is 5.81 Mb, revealing 5,673 coding sequences. It contains genes for cellulose-degradation and biosynthesis pathways of polyhydroxybutyrate (PHB) and 8 rRNA genes (5S, 16S, and 23S).

Violacein induces p44/42 mitogen-activated protein kinase-mediated solid tumor cell death and inhibits tumor cell migration.

Microbial secondary metabolites have emerged as alternative novel drugs for the treatment of human cancers. Violacein, a purple pigment produced by Chromobacterium violaceum, was investigated in the present study for its anti-tumor properties in tumor cell lines. Clinically applicable concentrations of violacein were demonstrated to inhibit the proliferative capacity of tumor cell lines according to a crystal violet proliferation assay. The underlying mechanism was the promotion of apoptotic cell death, as indicated by poly(ADP ribose) polymerase cleavage and p44/42 mitogen-activated protein kinase signaling determined by western blot analysis. Collectively, this provided mechanistic evidence that violacein elicits extracellular-signal regulated kinase-induced apoptosis via the intrinsic pathway. The anti-malignant properties of violacein in the present study were further demonstrated by its inhibitory effects on brain tumor cell migration, specifically glioblastomas, one of the most invasive and therapeutically resistant neoplasms in the clinic. Additionally, solid tumors examined in the present study displayed differential cellular responses and sensitivities to violacein as observed by morphologically induced cellular changes that contributed to its anti-migratory properties. In conclusion, violacein is a novel natural product with the potential to kill several types of human tumor cell lines, as well as prevent disease recurrence by antagonizing cellular processes that contribute to metastatic invasion.

Using size exclusion chromatography to monitor the synthesis of melanins from catecholamines

We have employed size exclusion chromatography (SEC) to the study of the auto- and Cu2+-mediated oxidation of the catecholamines, dopamine, epinephrine and norepinephrine, into melanins. We observed that, due to non-size exclusion-mediated effects, the catecholamines and some of the low molecular mass intermediates generated during the oxidation reactions, could be resolved from each other and from the high molecular mass pigment generated. Thus, SEC allowed us to monitor the disappearance of the catecholamine starting compounds, the appearance and subsequent disappearance of the low molecular mass chromophores generated in the initial phase of the reactions and the appearance of the high molecular mass melanins. In the process of this research, we observed that many, mostly anionic polysaccharides (PS), enhanced both the auto- and Cu2+-mediated oxidation of all three catecholamines. SEC analyses of reaction mixtures involving PS suggested that very high molecular mass aggregates between PS and melanins can be generated. In addition, SEC analysis allowed us to verify the efficiency of the dialysis purification process employed to obtain pure and dried melanin materials for cell-biological studies.

Polysaccharide-mediated synthesis of melanins from serotonin and other 5-hydroxy indoles

Aim: As a continuation of our research on the melanin formation from catecholamines, we studied the polysaccharide-mediated oxidation of serotonin and other 5-hydroxy indoles into melanin-like materials. As for the catecholamines, we observed that many polysaccharides promote the oxidation of such compounds, particularly in the presence of Cu2+. Methodology: The reactions were monitored using reverse phase-HPLC and size-exclusion chromatography techniques. Melanin-like materials were purified through dialysis and characterized using UV-Vis and Fourier transform IR spectroscopic techniques. Results: One such material, synthesized from chondroitin sulfate type A and serotonin in the presence of Cu2+ was found to affect the release of IL-1β and IL-6 cytokines from immune cells.