Contributions to our understanding of T cell physiology through unveiling the T cell proteome

Since the sequencing of the human genome was completed, attention has turned to examining the functionality of the molecular machinery, in particular of protein expression. Differential proteome analysis by two-dimensional electrophoresis has been adopted to study changes in T cell proteomes during T cell activation, and this work is increasing our understanding of the complexity of signals elicited across multiple pathways. The purpose of this review is to summarize the available evidence in the application of proteomic techniques and methodologies to understand T cell receptor activation from lipid raft and cytoskeletal rearrangements, through to signalling cascades, transcription factor modulation and changes in protein expression patterns. These include post-translational modifications, which are not encoded by the genome. © 2007 British Society for Immunology.

Physiological effects of oxidized phospholipids and their cellular signaling mechanisms in inflammation

Oxidized phospholipids, such as the products of the oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine by nonenzymatic radical attack, are known to be formed in a number of inflammatory diseases. Interest in the bioactivity and signaling functions of these compounds has increased enormously, with many studies using cultured immortalized and primary cells, tissues, and animals to understand their roles in disease pathology. Initially, oxidized phospholipids were viewed largely as culprits, in line with observations that they have proinflammatory effects, enhancing inflammatory cytokine production, cell adhesion and migration, proliferation, apoptosis, and necrosis, especially in vascular endothelial cells, macrophages, and smooth muscle cells. However, evidence has emerged that these compounds also have protective effects in some situations and cell types; a notable example is their ability to interfere with signaling by certain Toll-like receptors (TLRs) induced by microbial products that normally leads to inflammation. They also have protective effects via the stimulation of small GTPases and induce up-regulation of antioxidant enzymes and cytoskeletal rearrangements that improve endothelial barrier function. Oxidized phospholipids interact with several cellular receptors, including scavenger receptors, platelet-activating factor receptors, peroxisome proliferator-activated receptors, and TLRs. The various and sometimes contradictory effects that have been observed for oxidized phospholipids depend on their concentration, their specific structure, and the cell type investigated. Nevertheless, the underlying molecular mechanisms by which oxidized phospholipids exert their effects in various pathologies are similar. Although our understanding of the actions and mechanisms of these mediators has advanced substantially, many questions do remain about their precise interactions with components of cell signaling pathways.

Hsc70-induced changes in clathrin-auxilin cage structure suggest a role for clathrin light chains in cage disassembly

The molecular chaperone, Hsc70, together with its cofactor, auxilin, facilitates the ATP-dependent removal of clathrin during clathrin-mediated endocytosis in cells. We have used cryo-electron microscopy to determine the 3D structure of a complex of clathrin, auxilin401-910 and Hsc70 at pH 6 in the presence of ATP, frozen within 20 seconds of adding Hsc70 in order to visualize events that follow the binding of Hsc70 to clathrin and auxilin before clathrin disassembly. In this map,we observe density beneath the vertex of the cage that we attribute to bound Hsc70. This density emerges asymmetrically from the clathrin vertex, suggesting preferential binding by Hsc70 for one of the three possible sites at the vertex. Statistical comparison with a map of whole auxilin and clathrin previously published by us reveals the location of statistically significant differences which implicate involvement of clathrin light chains in structural rearrangements which occur after Hsc70 is recruited. Clathrin disassembly assays using light scattering suggest that loss of clathrin light chains reduces the efficiency with which auxilin facilitates this reaction. These data support a regulatory role for clathrin light chains in clathrin disassembly in addition to their established role in regulating clathrin assembly. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

The synthesis and properties of some hydrazines

A new synthetic method, applicable to the preparation of a wide range of hydrazine derivatives, is described. This involves the diborane reduction of a hydrazone, or, more conveniently, the reductive-condensation of a hydrazine and the appropriate aldehyde (or ketone). The method gives high yields and provides a particularly simple route to the relatively inaccessible 1,2-disubstituted hydrazines bearing a different group on each nitrogen. The new method has also been applied to the preparation of 1,2-disubstituted hydrazines with the same group on both nitrogens (via the azine), the very rare 1 ,2-disubstituted hydrazines bearing a tert-butyl group, trisubstituted hydrazines and monosubstituted hydrazines. Application of the reaction to the preparation of diaziridines has also been investigated. A mechanism for the reduction, supported by the isolation of a boron-containing intermediate, is suggested. Some limitations of the procedure are discussed. A general i.r. method of distinguishing the isomeric disubstituted hydrazines, as stable salts, has been developed. This has the advantages of speed and simplicity over previous methods. The mass spectra of a series of monosubstituted hydrazines, a series of 1,2-disubstituted hydrazines and some 1-benzoyl 2-alkylhydrazines have been examined in detail. The spectra are generally dominated byα -cleavage processes and the compounds show a variety of interesting rearrangement reactions. The mass spectra of some 1, 1-disubstituted hydrazines and some trisubstituted hydrazines have also been examined. Rearrangement processes occurring in the mass spectrum of tropylium fluoroborate have been examined. Similar rearrangements have been found in the spectrum of trityl fluoroborate and may be of general occurrence in the mass spectra of aromatic fluoroborates. Chemical shift values for some groups on hydrazine nitrogen are recorded and the results of tumour inhibitory tests on some hydrazines are also given.

Exploring the biological basis for Salmonella persistence in food manufacturing environments

The persistence of Salmonella spp. in low moisture foods is a challenge for the food industry as despite control strategies already in place, notable outbreaks still occur. The aim of this study was to characterise isolates of Salmonella, known to be persistent in the food manufacturing environment, by comparing their microbiological characteristics with a panel of matched clinical and veterinary isolates. The gross morphology of the challenge panel was phenotypically characterised in terms of cellular size, shape and motility. In all the parameters measured, the factory isolates were indistinguishable from the human, clinical and veterinary strains. Further detailed metabolic profiling was undertaken using the biolog Microbial ID system. Multivariate analysis of the metabolic microarray revealed differences in metabolism of the factory isolate of S.Montevideo, based on its upregulated ability to utilise glucose and the sugar alcohol groups. The remainder of the serotype-matched isolates were metabolically indistinguishable. Temperature and humidity are known to influence bacterial survival and through environmental monitoring experimental parameters were defined. The results revealed Salmonella survival on stainless steel was affected by environmental temperatures that may be experienced in a food processing environment; with higher survival rates (D25=35.4) at temperatures at 25°C and lower humidity levels of 15% RH, however a rapid decline in cell count (D10=3.4) with lower temperatures of 10°C and higher humidity of 70% RH. Several resident factories strains survived in higher numbers on stainless steel (D25=29.69) compared to serotype matched clinical and veterinary isolates (D25=22.98). Factory isolates of Salmonella did not show an enhanced growth rate in comparison to serotype matched solates grown in Luria broth, Nutrient broth and M9 minimal media indicating that as an independent factor, growth was unlikely to be a major factor driving Salmonella persistence. Using a live / dead stain coupled with fluorescence microscopy revealed that when no longer culturable, isolates of S.Schwarzengrund entered into a viable nonculturable state. The biofilm forming capacity of the panel was characterised and revealed that all were able to form biofilms. None of the factory isolates showed an enhanced capability to form biofilms in comparison to serotype-matched isolates. In disinfection studies, planktonic cells were more susceptible to disinfectants than cells in biofilm and all the disinfectants tested were successful in reducing bacterial load. Contact time was one of the most important factors for reducing bacterial populations in a biofilm. The genomes of eight strains were sequenced. At the nucleotide and amino acid level the food factory isolates were similar to those of isolates from other environments; no major genomic rearrangements were observed, supporting the conclusions of the phenotypic and metabolic analysis. In conclusion, having investigated a variety of morphological, biochemical and genomic factors, it is unlikely that the persistence of Salmonella in the food manufacturing environment is attributable to a single phenotypic, metabolic or genomic factor. Whilst a combination of microbiological factors may be involved it is also possible that strain persistence in the factory environment is a consequence of failure to apply established hygiene management principles.