Leigh A. Madden

CHAIN TERMINATION IN POLYHYDROXYALKANOATE SYNTHESIS: INVOLVEMENT OF EXOGENOUS HYDROXY-COMPOUNDS AS CHAIN TRANSFER AGENTS

We have identified a range of compounds which, when present during poly(3-hydroxybutyrate) [P(3HB)] accumulation by Ralstonia eutropha (reclassified from Alcaligenes eutrophus), can act as chain transfer agents in the chain termination step of polymerization. End-group analysis by 31P NMR of polymer derivatized with 2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane revealed that all these compounds were covalently linked to P(3HB) at the carboxyl terminus. All chain transfer agents possessed one or more hydroxyl groups, and glycerol was selected for further investigation. The number-average molecular mass (Mn) of P(3HB) produced by R. eutropha from glycerol was substantially lower than for polymer produced from glucose, and we identified two new end-group structures. These were attributed to a glycerol molecule bound to the P(3HB) chain via the primary or secondary hydroxyl groups. When a primary hydroxyl group of glycerol is involved in chain transfer, the end-group structure is in both [R] and [S] configurations, implying that chain transfer to glycerol is a random transesterification and that PHA synthase does not catalyse chain transfer. 3-Hydroxybutyric acid is the most probable chain transfer agent in vivo, with propagation and termination reactions involving transfer of the P(3HB) chain to enzyme-bound and free 3-hydroxybutyrate, respectively. Only carboxyl end-groups were detected in P(3HB) extracted from exponentially growing bacteria. It is proposed that a compound other than 3-hydroxybutyryl-CoA acts as a primer in the initiation of polymer synthesis.

Binding optimization through coordination chemistry: CXCR4 chemokine receptor antagonists from ultrarigid metal complexes

A new copper(II) containing bis-macrocyclic CXCR4 chemokine receptor antagonist is shown to have improved binding properties to the receptor protein in comparison to the drug AMD3100 (Plerixafor, Mozobil). The interaction of the metallodrug has been optimized by using ultrarigid chelator units that offer an equatorial site for coordination to the amino acid side chains of the protein. Binding competition assays with anti-CXCR4 antibodies show that the new compound stays bound longer and it has improved anti-HIV potency in vitro (EC(50) = 4.3 nM). X-ray structural studies using acetate as a model for carboxylate amino acid side chains indicate the nature of the coordination interaction.

Inducible heat shock protein 70 and its role in preconditioning and exercise

Heat shock proteins (Hsp) are well known to be expressed in response to a range of cellular stresses. They are known to convey protection against protein denaturation and a subsequent immediate stress. Inducible heat shock protein 70 (Hsp70) is among the most studied of these stress proteins and its role and function are discussed here in terms of thermal and in particular exercise preconditioning. Preconditioning has been shown to confer cellular protection via expression Hsp, which may be of benefit in preventing protein damage following subsequent periods of exercise. Many studies have used animal models to gather data on Hsp70 and these and the most recent human studies are discussed.

Viability of plant spore exine capsules for microencapsulation

Sporopollenin exine capsules (SECs) (outer exoskeletal wall of the spores of Lycopodium clavatum) were extracted and examined for their potential use as microcapsules. They were shown, by laser scanning confocal microscopy (LSCM), to be void of their inner contents. The removal of nitrogenous and other internal materials was supported by a combination of elemental and gravimetric analyses. Two different methods were investigated to encapsulate substances into SECs which were (i) mild passive migration of materials into the SECs and (ii) subjecting SECs and materials to a vacuum. A range of fluorescent dyes with different polarities were seen using LSCM to encapsulate efficiently into the SECs (up to 1 g.g−1). Relatively unstable materials with different polarities were encapsulated into the SECs: polyunsaturated oils, which are labile to oxidation, and the enzymes streptavidin-horseradish peroxidase (sHRP) and alkaline phosphatase (ALP). Irrespective of the encapsulation techniques employed no oxidation of the oils or denaturation of the enzymes was observed following their full recovery. This study gives the first indication of the viability of SECs to microencapsulate various potentially unstable materials without causing a detrimental effect.

Endothelial function and stress response after simulated dives to 18 msw breathing air or oxygen.

INTRODUCTION Decompression sickness is caused by gas bubbles released upon decompression. These bubbles have the potential to occlude blood vessels and damage the vascular endothelium. The aim of this study was to quantify damage to the vascular endothelium resulting from decompression by measuring endothelial microparticles (MP) and endothelial function. METHODS Five healthy male volunteers undertook a simulated (hyperbaric chamber) air dive and 1 wk later a second dive breathing 100% oxygen at 283 kPa (18 msw) for 60 min bottom time, decompressed with 5-min stops at 161 kPa (6 msw) and 131 kPa (3 msw). Endothelial function was tested pre- and postdive by reactive hyperemia peripheral artery tonometry (RH-PAT) and CD105 (Endoglin) positive MP were quantified by flow cytometry. Plasma E- and P-selectin, interleukin-6, and serum cortisol were also quantified. RESULTS RH-PAT showed a significantly decreased endothelial function post-decompression after breathing air when compared to oxygen (-0.33 +/- 0.27 vs. +0.18 +/- 0.14). CD105 MP pre- and postdive showed no change on the oxygen dive (460 +/- 370 to 360 +/- 163), however, they increased after breathing air (440 +/- 70 to 1306 +/- 359). There was no change in expression of CD105 on MP. Furthermore no changes were observed in plasma E- or P-selectin, IL-6, or serum cortisol. CONCLUSION From the data, at least in the time frame involved, there appears to be no detectable physiological/stress response to decompression, rather decompression from breathing air probably caused mechanical damage to the endothelium, resulting in both MP release and a reduction in endothelial function.

Gas bubbles may not be the underlying cause of decompression illness - The at-depth endothelial dysfunction hypothesis

Gas formed in tissues and the circulating blood due to decompression is thought to be a significant factor in the progression of decompression illness (DCI). DCI is a potential problem for a growing population of professional and recreational divers. We hypothesise that these gas bubbles are not the causative agent in progression of DCI, rather an exacerbating factor. Endothelial dysfunction caused by a temporary loss of haemostasis due to increased total oxidant status is postulated to be the cause in this at-depth endothelial dysfunction hypothesis. Breathing oxygen at any pressure increases the oxidant status in the circulation causing vasoconstriction; this increase can be prevented by antioxidants, such as Vitamin C, maintaining haemostasis and preventing activation of endothelium, leukocyte recruitment and subsequent localised inflammation. Bubbles have the potential to exacerbate the situation on decompression by damaging the vascular endothelium either through ischemia/reperfusion, physical contact with the endothelium or by an increase in shear stress. Furthermore, this damage may manifest itself in the release of endothelial membrane fragments (microparticles).

Microparticle-associated vascular adhesion molecule-1 and tissue factor follow a circadian rhythm in healthy human subjects

An increased risk of death or severe injury due to late-morning thrombotic events is well established. Tissue factor (TF) is the initiator of the coagulation cascade, and endothelial stresses, coupled with production of pro-coagulant microparticles (MP) are also important factors in loss of haemostasis. TF and vascular cell adhesion molecule-1 (VCAM-1) -positive cell microparticles were assessed periodically over a 24-hour (h) period in healthy human subjects to ascertain if they followed a circadian rhythm. Eleven healthy male subjects were assessed in a temperature-controlled environment with dietary intake consistent between subjects. Blood samples were taken every 4 h by venipuncture, and TF and VCAM-1 positive microparticles were quantified by flow cytometry. A significant circadian rhythm was observed in VCAM-1 MP (p=or<0.0001), and a trend was shown, although not statistically significant (p=0.065) in TF microparticles. A peak was observed at 9 a.m. for VCAM-1 positive MP, followed by a decrease and subsequent peak at 9 p.m. and a minimum at 5 a.m. TF-positive MP followed a strikingly similar trend in both variation and absolute numbers with a delay. A circadian rhythm was observed in VCAM-1 and less so TF-positive MP. This has significant implications in terms of the well known increased risk of cardiovascular thrombotic events matching this data. To our knowledge this is the first such report of quantified measurements of these MP over a 24-h period and the only measurement of a 24-h variation of in-vivo blood-borne TF.

Tumour and microparticle tissue factor expression and cancer thrombosis

Cancer is frequently complicated by venous thromboembolic events (VTE), which pose a significant health burden due to the associated high morbidity and mortality rates, yet the exact details of the pathophysiological mechanisms underlying their development are yet to be fully elucidated. Tissue factor (TF), the primary initiator of coagulation, is often overexpressed in malignancy and as such is a prime candidate in predicting the hypercoagulable state. Further exploration of this potential role has identified increases in the number of TF-expressing microparticles (MP) in the circulation of cancer patients, in particular in those known to have high incidences of thromboembolic complications. The risk of VTE in cancer is found to be further elevated by chemotherapy. Chemotherapy may, in eliciting cancer cell apoptosis, result in an increase in release of circulating procoagulant MP. We discuss a potential role of elevated tumour TF expression and increased circulating TF-positive MP in predicting VTE risk.

Pancreatic cancer cell and microparticle procoagulant surface characterization: involvement of membrane-expressed tissue factor, phosphatidylserine and phosphatidylethanolamine.

Advanced pancreatic cancer is associated with a high risk of patients developing venous thromboembolism. This increased risk is thought to be tumour-driven and associated with tissue factor (TF) and microparticles. The aim of this study was to investigate the role of TF and phospholipid expression in the procoagulant properties of pancreatic cell lines and microparticles. Pancreatic cancer cell lines (MIA-PaCa-2, ASPC-1 and CFPAC-1) were assessed for expression of TF and microparticle release. Procoagulant potential was determined by a prothrombin time assay. Cell surface expression of TF was highest in CFPAC-1, with low expression on ASPC-1 and little/no expression on MIA-PaCa-2. Clotting time (CT) was cell number and TF-dependent (P < 0.001). Blocking of TF resulted in slower CT for CFPAC-1 and ASPC1 and prevented clotting in MIA-PaCa-2. Microparticles were shown to be procoagulant and the majority of procoagulant potential could be removed by passing cell-free media through a 0.1 &mgr;m filter. A dose-dependent CT was observed in both ASPC-1 and CFPAC-1 cell-free media. Furthermore, addition of duramycin prevented microparticle-supported coagulation. The data presented suggest a key role for cell and microparticle surface-expressed TF and phospholipids in coagulation and highlight duramycin-mediated disruption of clotting.

High-polyphenol chocolate reduces endothelial dysfunction and oxidative stress during acute transient hyperglycaemia in Type 2 diabetes: a pilot randomized controlled trial.

To investigate the effects of high‐polyphenol chocolate upon endothelial function and oxidative stress in Type 2 diabetes mellitus during acute transient hyperglycaemia induced following a 75‐g oral glucose challenge.