Platelet 12-lipoxygenase activation via glycoprotein VI - Involvement of multiple signaling pathways in agonist control of H(P)ETE synthesis

Lipoxygenases (LOX) contribute to vascular disease and inflammation through generation of bioactive lipids, including 12-hydro(pero)xyeicosatetraenoic acid (12-H(P)ETE). The physiological mechanisms that acutely control LOX product generation in mammalian cells are uncharacterized. Human platelets that contain a 12-LOX isoform (p12-LOX) were used to define pathways that activate H( P) ETE synthesis in the vasculature. Collagen and collagen-related peptide (CRP) (1 to 10 mug/mL) acutely induced platelet 12-H(P)ETE synthesis. This implicated the collagen receptor glycoprotein VI ( GPVI), which signals via the immunoreceptor-based activatory motif (ITAM)-containing FcRgamma chain. Conversely, thrombin only activated at high concentrations (> 0.2 U/mL), whereas U46619 and ADP alone were ineffective. Collagen or CRP-stimulated 12-H( P) ETE generation was inhibited by staurosporine, PP2, wortmannin, BAPTA/AM, EGTA, and L-655238, implicating src-tyrosine kinases, PI3-kinase, Ca2+ mobilization, and p12-LOX translocation. In contrast, protein kinase C (PKC) inhibition potentiated 12-H( P) ETE generation. Finally, activation of the immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing platelet endothelial cell adhesion molecule (PECAM-1) inhibited p12-LOX product generation. This study characterizes a receptor-dependent pathway for 12-H(P) ETE synthesis via the collagen receptor GPVI, which is negatively regulated by PECAM-1 and PKC, and demonstrates a novel link between immune receptor signaling and lipid mediator generation in the vasculature.

Novel biomimetic and bioactive silicones

The features and potential applications of a novel silicone for constructing or coating medical devices are described. The platform technology that has been developed reportedly overcomes inherent problems with existing silicone devices and allows drug delivery from devices employing the material.

A critical evaluation of Raman spectroscopy for the analysis of lipids: fatty acid methyl esters.

The work presented here is aimed at determining the potential and limitations of Raman spectroscopy for fat analysis by carrying out a systematic investigation of C-4-C-24 FAME. These provide a simple, well-characterized set of compounds in which the effect of making incremental changes can be studied over a wide range of chain lengths and degrees of unsaturation. The effect of temperature on the spectra was investigated over much larger ranges than would normally be encountered in real analytical measurements. It was found that for liquid FAME the best internal standard band was the carbonyl stretching vibration nu(C = O), whose position is affected by changes in sample chain length and physical state; in the samples studied here, it was found to lie between 1729 and 1748 cm(-1). Further, molar unsaturation could be correlated with the ratio of the nu(C = O) to either nu(C = C) or delta(H-C = ) with R-2 > 0.995. Chain length was correlated with the delta(CH2)(tw)/nu(C = O) ratio, (where "tw" indicates twisting) but separate plots for odd- and even-numbered carbon chains were necessary to obtain R-2 > 0.99 for liquid samples. Combining the odd- ani even-numbered carbon chain data in a single plot reduced the correlation to R-2 = 0.94-0.96, depending on the band ratios used. For molal unsaturation the band ratio that correlated linearly with unsaturation (R-2 > 0.99) was nu(C = C)/delta(CH2)(SC) (where "sc" indicates scissoring). Other band ratios show much more complex behavior with changes in chemical and physical structure. This complex behavior results from the fact that the bands do not arise from simple vibrations of small, discrete regions of the molecules but are due to complex motions of large sections of the FAME so that making incremental changes in structure does not necessarily lead to simple incremental changes in spectra.

Physicochemical characterization of bioactive polyacrylic acid organogels as potential antimicrobial implants for the buccal cavity

This study describes the formulation and physicochemical characterization of poly(acrylic acid) (PAA) organogels, designed as bioactive implants for improved treatment of infectious diseases of the oral cavity. Organogels were formulated containing a range of concentrations of PAA (3-10% w/w) and metronidazole (2 or 5% w/w, representing a model antimicrobial agent) in different nonaqueous solvents, namely, glycerol (Gly), polyethylene glycol (PEG 400), or propylene glycol (PG). Characterization of the organogels was performed using flow rheometry, compressional analysis, oscillatory rheometry, in vitro mucoadhesion, moisture uptake, and drug release, methods that provide information pertaining to the nonclinical and clinical use of these systems. Increasing the concentration of PAA significantly increased the consistency, compressibility, storage modulus, loss modulus, dynamic viscosity, mucoadhesion, and the rate of drug release. These observations may be accredited to enhanced molecular polymer entanglement. In addition, the choice of solvent directly affected the physicochemical parameters of the organogels, with noticeable differences observed between the three solvents examined. These differences were accredited to the nature of the interaction of PAA with each solvent and, importantly, the density of the resultant physical cross-links. Good correlation was observed between the viscoelastic properties and drug release, with the exception of glycerol-based formulations containing 5 and 10% w/w PAA. This disparity was due to excessive swelling during the dissolution analysis. Ideally, formulations should exhibit controlled drug release, high viscoelasticity, and mucoadhesion, but should flow under minimal stresses. Based on these criteria, PEG 400-based organogels composed of 5% or 10% w/w PAA exhibited suitable physicochemical properties and are suggested to be a potentially interesting strategy for use as bioactive implants designed for use in the oral cavity. © 2008 American Chemical Society.

Production of bioactive cellulose films reconstituted from ionic liquids

A new method for introducing enzymes into cellulosic matrixes which can be formed into membranes, films, or beads has been developed using a cellulose-in-ionic-liquid dissolution and regeneration process. Initial results on the formation of thin cellulose films incorporating dispersed laccase indicate that active enzyme-encapsulated films can be prepared using this methodology and that precoating the enzyme with a second. hydrophobic ionic liquid prior to dispersion in the cellulose/ionic liquid solution can provide an increase in enzyme activity relative to that of untreated films, presumably by providing a stabilizing microenvironment for the enzyme.

Helokinestatins: A novel family of bioactive peptides with drug-lead potential fromthe venomof the Gila Monster (Heloderma suspectum)

Venom of the Gila Monster (Heloderma suspectum) has proven to be an unlikely source of lead compounds (exendins) for the development of new injectable peptide therapeutics for the treatment of Type 2 diabetes. However, no systematic searches for new classes of bioactive peptides in lizard venom have appeared until recently. Here we describe the discovery of a new class of peptides – the helokinestatins – from H. suspectum venom, their structural characterisation and that of their biosynthetic precursors from cloned cDNA. In addition, we have subjected members of the family to preliminary pharmacological characterisation. Helokinestatins 1–6 are a family of proline-rich peptides containing 10–15 amino acid residues terminating in a common -Pro-Arg.OH motif. They are encoded in tandem within two virtually identical biosynthetic precursors of 177 and 178 amino acid residues, differing by only a single Pro residue. Each precursor also encodes a single copy of a C-type natriuretic peptide located at the C-terminus. Synthetic replicates of all helokinestatins were shown to be devoid of any direct action on the smooth muscle of rat tail artery but were found to be potent inhibitors of bradykinin-induced relaxation in this preparation in a manner that is suggestive of a non-competitive mechanism. Helokinestatin-3 (VPPPPLQMPLIPR) and helokinestatin-5 (VPPPLQMPLIPR) were found to be most potent in this respect causing almost complete inhibition of bradykinin-induced relaxation. Helokinestatins and BPPs may have a shared evolutionary history but the former do not inhibit ACE. The bradykinin inhibitory potential of helokinestatins may be exploited in the local control of chronic inflammation.