11 research outputs found
Pentosan polysulfate promotes proliferation and chondrogenic differentiation of adult human bone marrow-derived mesenchymal precursor cells
No full textExtent: 17p.Introduction: This study was undertaken to determine whether the anti-osteoarthritis drug pentosan polysulfate (PPS) influenced mesenchymal precursor cell (MPC) proliferation and differentiation. Methods: Human MPCs were maintained in monolayer, pellet or micromass cultures (MMC) for up to 10 days with PPS at concentrations of 0 to 20 μg/ml. MPC viability and proliferation was assessed using the WST-1 assay and 3H-thymidine incorporation into DNA, while apoptosis was monitored by flow cytometry. Proteoglycan (PG) biosynthesis was determined by 35SO4 2- incorporation and staining with Alcian blue. Proteoglycan and collagen type I and collagen type II deposition in pellet cultures was also examined by Toluidine blue and immunohistochemical staining, respectively. The production of hyaluronan (HA) by MPCs in MMC was assessed by ELISA. The relative outcome of PPS, HA, heparin or dextran sulfate (DS) on PG synthesis was compared in 5-day MMC. Gene expression of MPCs in 7-day and 10-day MMC was examined using real-time PCR. MPC differentiation was investigated by co-culturing with PPS in osteogenic or adipogenic inductive culture media for 28 days. Results: Significant MPC proliferation was evident by day 3 at PPS concentrations of 1 to 5 μg/ml (P < 0.01). In the presence of 1 to 10 μg/ml PPS, a 38% reduction in IL-4/IFNγ-induced MPC apoptosis was observed. In 5-day MMC, 130% stimulation of PG synthesis occurred at 2.5 μg/ml PPS (P < 0.0001), while 5.0 μg/ml PPS achieved maximal stimulation in the 7-day and 10-day cultures (P < 0.05). HA and DS at ≥ 5 μg/ml inhibited PG synthesis (P < 0.05) in 5-day MMC. Collagen type II deposition by MMC was significant at ≥ 0.5 μg/ml PPS (P < 0.001 to 0.05). In MPC-PPS pellet cultures, more PG, collagen type II but less collagen type I was deposited than in controls. Real-time PCR results were consistent with the protein data. At 5 and 10 μg/ml PPS, MPC osteogenic differentiation was suppressed (P < 0.01). Conclusions: This is the first study to demonstrate that PPS promotes MPC proliferation and chondrogenesis, offering new strategies for cartilage regeneration and repair in osteoarthritic joints.Peter Ghosh, Jiehua Wu, Susan Shimmon, Andrew CW Zannettino, Stan Gronthos and Silviu Itesc
Pethidinic acid: Corroboration of a doctors denial of pethidine re-use
No full textPethidine (meperidine), a synthetic opiate, formally used as an analgesic in surgery and obstetrics, has been an abused drug of choice for some doctors. A case is presented in which a doctor, who previously admitted to using pethidine, was suspected of re-using, following a second positive urine test. A laboratory had reported the presence of pethidine in the doctors urine; however, the doctor denied re-use. The norpethidine (normeperidine) metabolite, normally found in urine, had not been detected, raising concern over the laboratorys conclusion and necessitating an independent investigation. Because the major metabolite of pethidine is pethidinic acid (meperidinic acid), accounting for approximately 40 of the excreted dose, its presence or absence were deemed to be important criteria in interpreting the laboratory result. Pethidinic acid was synthesized by alkaline hydrolysis of pethidine and used as a control. Urine samples from a patient receiving pethidine for pain, from the previous pethidine use of the doctor, and the urine under question plus the control were analyzed for the presence of pethidinic acid using electrospray mass spectrometry. Pethidinic acid was found in all samples except the one under dispute. The absence of pethidinic acid appeared to corroborate the doctors denial of re-use. © The Author [2013]. Published by Oxford University Press. All rights reserved
Biosensor analysis of the molecular interactions of pentosan polysulfate and of sulfated glycosaminoglycans with immobilized elastase, hyaluronidase and lysozyme using surface plasmon resonance (SPR) technology
No full textA System for Assessing Dual Action Modulators of Glycine Transporters and Glycine Receptors
No full textReduced inhibitory glycinergic neurotransmission is implicated in a number of neurological conditions such as neuropathic pain, schizophrenia, epilepsy and hyperekplexia. Restoring glycinergic signalling may be an effective method of treating these pathologies. Glycine transporters (GlyTs) control synaptic and extra-synaptic glycine concentrations and slowing the reuptake of glycine using specific GlyT inhibitors will increase glycine extracellular concentrations and increase glycine receptor (GlyR) activation. Glycinergic neurotransmission can also be improved through positive allosteric modulation (PAM) of GlyRs. Despite efforts to manipulate this synapse, no therapeutics currently target it. We propose that dual action modulators of both GlyTs and GlyRs may show greater therapeutic potential than those targeting individual proteins. To show this, we have characterized a co-expression system in Xenopus laevis oocytes consisting of GlyT1 or GlyT2 co-expressed with GlyRα1. We use two electrode voltage clamp recording techniques to measure the impact of GlyTs on GlyRs and the effects of modulators of these proteins. We show that increases in GlyT density in close proximity to GlyRs diminish receptor currents. Reductions in GlyR mediated currents are not observed when non-transportable GlyR agonists are applied or when Na+ is not available. GlyTs reduce glycine concentrations across different concentration ranges, corresponding with their ion-coupling stoichiometry, and full receptor currents can be restored when GlyTs are blocked with selective inhibitors. We show that partial inhibition of GlyT2 and modest GlyRα1 potentiation using a dual action compound, is as useful in restoring GlyR currents as a full and potent single target GlyT2 inhibitor or single target GlyRα1 PAM. The co-expression system developed in this study will provide a robust means for assessing the likely impact of GlyR PAMs and GlyT inhibitors on glycine neurotransmission
Influence of Bound versus Non-Bound Stabilizing Molecules on the Thermal Stability of Gold Nanoparticles
No full text
The allosteric inhibition of glycine transporter 2 by bioactive lipid analgesics is controlled by penetration into a deep lipid cavity
No full textThe role of lipids in modulating membrane protein function is an emerging and rapidly growing area of research. The rational design of lipids that target membrane proteins for the treatment of pathological conditions is a novel extension in this field and provides a step forward in our understanding of membrane transporters. Bioactive lipids show considerable promise as analgesics for the treatment of chronic pain and bind to a high-affinity allosteric binding site on the human glycine transporter 2 (GlyT2 or SLC6A5). Here we use a combination of medicinal chemistry, electrophysiology, and computational modelling to develop a rational structure activity relationship for lipid inhibitors and demonstrate the key role of the lipid tail interactions for GlyT2 inhibition. Specifically, we examine how lipid inhibitor head group stereochemistry, tail length and double bond position promote enhanced inhibition. Overall, the L-stereoisomer is generally a better inhibitor than the D-stereoisomer, longer tail length correlates with greater potency, and the position of the double bond influences the activity of the inhibitor. We propose that the binding of the lipid inhibitor deep into the allosteric binding pocket is critical for inhibition. Furthermore, this provides insight into the mechanism of inhibition of GlyT2 and highlights how lipids can modulate the activity of membrane proteins by binding to cavities between helices. The principles identified in this work have broader implications for the development of a larger class of compounds that could target SLC6 transporters for disease treatment
Pentosan polysulfate binds to STRO-1+ mesenchymal progenitor cells, is internalized, and modifies gene expression: a novel approach of pre-programing stem cells for therapeutic application requiring their chondrogenesis
No full textAbstract Background The pharmaceutical agent pentosan polysulfate (PPS) is known to induce proliferation and chondrogenesis of mesenchymal progenitor cells (MPCs) in vitro and in vivo. However, the mechanism(s) of action of PPS in mediating these effects remains unresolved. In the present report we address this issue by investigating the binding and uptake of PPS by MPCs and monitoring gene expression and proteoglycan biosynthesis before and after the cells had been exposed to limited concentrations of PPS and then re-established in culture in the absence of the drug (MPC priming). Methods Immuno-selected STRO-1+ mesenchymal progenitor stem cells (MPCs) were prepared from human bone marrow aspirates and established in culture. The kinetics of uptake, shedding, and internalization of PPS by MPCs was determined by monitoring the concentration-dependent loss of PPS media concentrations using an enzyme-linked immunosorbent assay (ELISA) and the uptake of fluorescein isothiocyanate (FITC)-labelled PPS by MPCs. The proliferation of MPCs, following pre-incubation and removal of PPS (priming), was assessed using the Wst-8 assay method, and proteoglycan synthesis was determined by the incorporation of 35SO4 into their sulphated glycosaminoglycans. The changes in expression of MPC-related cell surface antigens of non-primed and PPS-primed MPCs from three donors was determined using flow cytometry. RNA sequencing of RNA isolated from non-primed and PPS-primed MPCs from the same donors was undertaken to identify the genes altered by the PPS priming protocol. Results The kinetic studies indicated that, in culture, PPS rapidly binds to MPC surface receptors, followed by internalisation and localization within the nucleus of the cells. Following PPS-priming of MPCs and a further 48 h of culture, both cell proliferation and proteoglycan synthesis were enhanced. Reduced expression of MPC-related cell surface antigen expression was promoted by the PPS priming, and RNA sequencing analysis revealed changes in the expression of 42 genes. Conclusion This study has shown that priming of MPCs with low concentrations of PPS enhanced chondrogenesis and MPC proliferation by modifying their characteristic basal gene and protein expression. These findings offer a novel approach to re-programming mesenchymal stem cells for clinical indications which require the repair or regeneration of cartilaginous tissues such as in osteoarthritis and degenerative disc disease
Novel Phenylene Lipids That Are Positive Allosteric Modulators of Glycine Receptors and Inhibitors of Glycine Transporter 2
No full textChronic pain is a complex condition that remains resistant
to current
therapeutics. We previously synthesized a series of N-acyl amino acids (NAAAs) that inhibit the glycine transporter, GlyT2,
some of which are also positive allosteric modulators of glycine receptors
(GlyRs). In this study, we have synthesized a library of NAAAs that
contain a phenylene ring within the acyl tail with the objective of
improving efficacy at both GlyT2 and GlyRs and also identifying compounds
that are efficacious as dual-acting modulators to enhance glycine
neurotransmission. The most efficacious positive allosteric modulator
of GlyRs was 2-[8-(2-octylphenyl)octanoylamino]acetic acid (8–8
OPGly) which potentiates the EC5 for glycine activation
of GlyRα1 by 1500% with an EC50 of 664
nM. Phenylene-containing NAAAs with a lysine headgroup were the most
potent inhibitors of GlyT2 with (2S)-6-amino-2-[8-(3-octylphenyl)octanoylamino]hexanoic
acid (8–8 MPLys) inhibiting GlyT2 with an IC50 of
32 nM. The optimal modulator across both proteins was (2S)-6-amino-2-[8-(2-octylphenyl)octanoylamino]hexanoic acid (8–8
OPLys), which inhibits GlyT2 with an IC50 of 192 nM and
potentiates GlyRs by up to 335% at 1 μM. When tested in a dual
GlyT2/GlyRα1 expression system, 8–8 OPLys
caused the greatest reductions in the EC50 for glycine.
This suggests that the synergistic effects of a dual-acting modulator
cause greater enhancements in glycinergic activity compared to single-target
modulators and may provide an alternate approach to the development
of new non-opioid analgesics for the treatment of chronic pain
