Synthetic ceramides inhibit CD36 expression in U937 cells through a reduction in cytosolic peroxide

Ceramide (a sphingolipid) and reactive oxygen species (ROS) are each partly responsible for the intracellular signal transduction of a variety of physiological, pharmacological or environmental agents. It has been reported that synthesis of ceramide and ROS are intimately linked, and show reciprocal regulation. The levels of ceramide are reported to be elevated in atherosclerotic plaques providing circumstantial evidence for a pro-atherogenic role for ceramide. Indeed, LDL may be important sources of ceramide from sphingomyelin, where it promotes LDL aggregation. Using synthetic, short chain ceramides to mimic the cellular responses to fluctuations in natural endogenous ceramides, we have investigated ceramide effects on both intracellular redox state (as glutathione and ROS) and redox-sensitive gene expression, specifically the scavenger receptor CD36 (using RT-PCR and flow cytometry), in U937 monocytes and macrophages. We describe that the principal redox altering properties of ceramide are to lower cytosolic peroxide and to increase mitochondrial ROS formation, where growth arrest of U937 monocytes is also observed. In addition, cellular glutathione was depleted, which was independent of an increase in glutathione peroxidase activity. Examination of the effects of ceramide on stress induced CD36 expression in macrophages, revealed a dose dependent reduction in CD36 mRNA and protein levels, which was mimicked by N-acetyl cysteine. Taken together, these data suggest that ceramides differentially affect ROS within different cellular compartments, and that loss of cytosolic peroxide inhibits expression of the redox sensitive gene, CD36. This may attenuate both the uptake of oxidised LDL and the interaction of HDL with macrophages. The resulting sequelae in vivo remain to be determined.

Synthetic ceramides induce growth arrest or apoptosis by altering cellular redox status

Reactive oxygen species (ROS) and ceramide are each partly responsible for the signal transduction of a variety of extracellular agents. Furthermore, the application of synthetic, short-chain ceramides mimics the cellular responses to these extracellular agents. However, the significance of ROS involvement in ceramide signaling pathways is poorly understood. Here we describe that the (cellular responses to C2-/C6-ceramide of growth arrest in U937 monocytes and apoptosis in Jurkat T-cells are preceded by a rise in mitochondrial peroxide production. In Jurkat T-cells, this is associated with a large time- and dose-dependent loss of cellular glutathione. However, in U937 monocytes, glutathione loss is transient. Differences in the magnitude and kinetics of this alteration in cellular redox state associate with discrete outcomes, namely growth arrest or apoptosis. © 2002 Elsevier Science (USA). All rights reserved.

Synthetic molecular mimics of naturally occurring cyclopentenones exhibit antifungal activity towards pathogenic fungi

The naturally occurring reactive electrophilic species 12-oxo-phytodienoic acid (12-oxo-PDA) is a potent antifungal agent, whereas the plant growth regulator jasmonic acid, which is synthesized from 12-oxo-PDA, is ineffective. To address what structural features of the molecule endow it with antifungal activity, we synthesized a series of molecular mimics of 12-oxo-PDA varying in the length of the alkyl chain at its C-4 ring position. The octyl analogue (4-octyl cyclopentenone) was the most effective at suppressing spore germination and subsequent mycelial growth of a range of fungal pathogens and was particularly effective against Cladosporium herbarum and Botrytis cinerea, with minimum fungicidal concentrations in the range 100-200 µM. Introduction of a carboxyl group to the end of the chain, mimicking natural fatty acids, markedly reduced antifungal efficacy. Electrolyte leakage, indicative of membrane perturbation, was evident in both C. herbarum and B. cinerea exposed to 4-octyl cyclopentenone. Lipid composition analysis of the fungal spores revealed that those species with a high oil content, namely Fusarium oxysporum and Alternaria brassicicola, were less sensitive to 4-octyl cyclopentenone. The comparable hydrophobicity of 4-octyl cyclopentenone and 12-oxo-PDA accounts for the similar spore suppression activity of these two compounds. The relative ease of synthesis of 4-octyl cyclopentenone makes it an attractive compound for potential use as an antifungal agent. © 2011 SGM.

A novel method for studying the cortical processing of human swallowing using Synthetic Aperture Magnetometry(SAM)

Background/Aims: Positron emission tomography has been applied to study cortical activation during human swallowing, but employs radio-isotopes precluding repeated experiments and has to be performed supine, making the task of swallowing difficult. Here we now describe Synthetic Aperture Magnetometry (SAM) as a novel method of localising and imaging the brain's neuronal activity from magnetoencephalographic (MEG) signals to study the cortical processing of human volitional swallowing in the more physiological prone position. Methods: In 3 healthy male volunteers (age 28–36), 151-channel whole cortex MEG (Omega-151, CTF Systems Inc.) was recorded whilst seated during the conditions of repeated volitional wet swallowing (5mls boluses at 0.2Hz) or rest. SAM analysis was then performed using varying spatial filters (5–60Hz) before co-registration with individual MRI brain images. Activation areas were then identified using standard sterotactic space neuro-anatomical maps. In one subject repeat studies were performed to confirm the initial study findings. Results: In all subjects, cortical activation maps for swallowing could be generated using SAM, the strongest activations being seen with 10–20Hz filter settings. The main cortical activations associated with swallowing were in: sensorimotor cortex (BA 3,4), insular cortex and lateral premotor cortex (BA 6,8). Of relevance, each cortical region displayed consistent inter-hemispheric asymmetry, to one or other hemisphere, this being different for each region and for each subject. Intra-subject comparisons of activation localisation and asymmetry showed impressive reproducibility. Conclusion: SAM analysis using MEG is an accurate, repeatable, and reproducible method for studying the brain processing of human swallowing in a more physiological manner and provides novel opportunities for future studies of the brain-gut axis in health and disease.

Biocomposites of non-crosslinked natural and synthetic polymers

Biocomposite films comprising a non-crosslinked, natural polymer (collagen) and a synthetic polymer, poly(var epsilon-caprolactone) (PCL), have been produced by impregnation of lyophilised collagen mats with a solution of PCL in dichloromethane followed by solvent evaporation. This approach avoids the toxicity problems associated with chemical crosslinking. Distinct changes in film morphology, from continuous surface coating to open porous format, were achieved by variation of processing parameters such as collagen:PCL ratio and the weight of the starting lyophilised collagen mat. Collagenase digestion indicated that the collagen content of 1:4 and 1:8 collagen:PCL biocomposites was almost totally accessible for enzymatic digestion indicating a high degree of collagen exposure for interaction with other ECM proteins or cells contacting the biomaterial surface. Much reduced collagen exposure (around 50%) was measured for the 1:20 collagen:PCL materials. These findings were consistent with the SEM examination of collagen:PCL biocomposites which revealed a highly porous morphology for the 1:4 and 1:8 blends but virtually complete coverage of the collagen component by PCL in the1:20 samples. Investigations of the attachment and spreading characteristics of human osteoblast (HOB) cells on PCL films and collagen:PCL materials respectively, indicated that HOB cells poorly recognised PCL but attachment and spreading were much improved on the biocomposites. The non-chemically crosslinked, collagen:PCL biocomposites described are expected to provide a useful addition to the range of biomaterials and matrix systems for tissue engineering.

Transglutaminase mediated grafting of silk proteins onto wool fabrics leading to improved physical and mechanical properties

Transglutaminases have the ability to incorporate primary amines and to graft peptides (containing glutamine or lysine residues) into proteins. These properties enable transglutaminases to be used in the grafting of a range of compounds including peptides and/or proteins onto wool fibres, altering their functionality. In this paper we investigated the transglutaminase mediated grafting of silk proteins into wool and its effect on wool properties. A commercial hydrolysed silk preparation was compared with silk sericin. The silk sericin protein was labelled with a fluorescent probe which was used to demonstrate the efficiency of the TGase grafting of such proteins into wool fibres. The TGase mediated grafting of these proteins led to a significant effect on the properties of wool yarn and fabric, resulting in increased bursting strength, as well as reduced levels of felting shrinkage and improved fabric softness. Also observed was an accumulation of deposits on the surface of the treated wool fibres when monitored by SEM and alterations in the thermal behaviour of the modified fibres, in particular for mTGase/sericin treated fibres which, with the confocal studies, corroborate the physical changes observed on the treated wool fabric. © 2006 Elsevier Inc. All rights reserved.

Transglutaminase treatment of wool fabrics leads to resistance to detergent damage

Biological detergents are now routinely used in domestic laundry because the enzymes they contain provide the added benefit of low temperature washes with improved cleaning performance. One of the key enzymes found in these detergents are proteases, which if exposed to natural protein fibres such as wool or silk can cause irreversible damage, leading to loss of fabric strength, shape and poor colour fastness. Transglutaminases (TGases) are protein cross-linking enzymes capable of adding tensile strength to wool proteins, and as a consequence are capable of remediating the damage caused by previous chemical treatments, and more importantly, by proteases. In this paper we treated dyed wool fabric with TGase and then washed the fabric with biological and non-biological detergents to investigate whether TGases would protect wool garments from damage by the undue use of biological detergents in domestic laundry. We demonstrate using different cycles of detergent washes containing biological and non-biological detergents and different TGase treatments, that wool fabric treated previously with TGase release less dye into the washing liquor and in addition maintain fabric strength at levels greater than the washed controls. As a consequence, wool garments previously treated with TGase are likely to have increased resistance to domestic washing and thus provide increased longevity. © 2005 Elsevier B.V. All rights reserved.