Interleukin-2 signalling is modulated by a labile disulfide bond in the CD132 chain of its receptor

Certain disulfide bonds present in leucocyte membrane proteins are labile and can be reduced in inflammation. This can cause structural changes that result in downstream functional effects, for example, in integrin activation. Recent studies have shown that a wide range of membrane proteins have labile disulfide bonds including CD132, the common gamma chain of the receptors for several cytokines including interleukin-2 and interleukin-4 (IL-2 and IL-4). The Cys183–Cys232 disulfide bond in mouse CD132 is susceptible to reduction by enzymes such as thioredoxin (TRX), gamma interferon-inducible lysosomal thiolreductase and protein disulfide isomerase, which are commonly secreted during immune activation. The Cys183–Cys232 disulfide bond is also reduced in an in vivo lipopolysaccharide (LPS)-induced acute model of inflammation. Conditions that lead to the reduction of the Cys183–Cys232 disulfide bond in CD132 inhibit proliferation of an IL-2-dependent T cell clone and concomitant inhibition of the STAT-5 signalling pathway. The same reducing conditions had no effect on the proliferation of an IL-2-independent T cell clone, nor did they reduce disulfide bonds in IL-2 itself. We postulate that reduction of the Cys183–Cys232 disulfide in CD132 inhibits IL-2 binding to the receptor complex. Published data show that the Cys183–Cys232 disulfide bond is exposed at the surface of CD132 and in close contact with IL-2 and IL-4 in their respective receptor complexes. In addition, mutants in these Cys residues in human CD132 lead to immunodeficiency and loss of IL-2 binding. These results have wider implications for the regulation of cytokine receptors in general, as their activity can be modulated by a ‘redox regulator’ mechanism caused by the changes in the redox environment that occur during inflammation and activation of the immune system

Labile disulfide bonds are common at the leucocyte cell surface

Redox conditions change in events such as immune and platelet activation, and during viral infection, but the biochemical consequences are not well characterized. There is evidence that some disulfide bonds in membrane proteins are labile while others that are probably structurally important are not exposed at the protein surface. We have developed a proteomic/mass spectrometry method to screen for and identify non-structural, redox-labile disulfide bonds in leucocyte cell-surface proteins. These labile disulfide bonds are common, with several classes of proteins being identified and around 30 membrane proteins regularly identified under different reducing conditions including using enzymes such as thioredoxin. The proteins identified include integrins, receptors, transporters and cell–cell recognition proteins. In many cases, at least one cysteine residue was identified by mass spectrometry as being modified by the reduction process. In some cases, functional changes are predicted (e.g. in integrins and cytokine receptors) but the scale of molecular changes in membrane proteins observed suggests that widespread effects are likely on many different types of proteins including enzymes, adhesion proteins and transporters. The results imply that membrane protein activity is being modulated by a ‘redox regulator’ mechanism

Observation of the dynamic Jahn-Teller effect in the excited states of nitrogen-vacancy centers in diamond

The optical transition linewidth and emission polarization of single nitrogen-vacancy (NV) centers are measured from 5 K to room temperature. Inter-excited state population relaxation is shown to broaden the zero-phonon line and both the relaxation and linewidth are found to follow a T^5 dependence for T up to 100 K. This dependence indicates that the dynamic Jahn-Teller effect is the dominant dephasing mechanism for the NV optical transitions at low temperatures

Analysis of leukocyte membrane protein interactions using protein microarrays

BACKGROUND: Protein microarrays represent an emerging class of proteomic tools to investigate multiple protein-protein interactions in parallel. A sufficient proportion of immobilized proteins must maintain an active conformation and an orientation that allows for the sensitive and specific detection of antibody and ligand binding. In order to establish protein array technology for the characterization of the weak interactions between leukocyte membrane proteins, we selected the human leukocyte membrane protein CD200 (OX2) and its cell surface receptor (hCD200R) as a model system. As antibody-antigen reactions are generally of higher affinity than receptor-ligand binding, we first analyzed the reactivity of monoclonal antibodies (mAb) to normal and mutant forms of immobilized CD200R. RESULTS: Fluorescently labelled mAb DX147, DX136 and OX108 were specifically reactive with immobilized recombinant hCD200R extracellular region, over a range of 0.1–40 μg ml(-1 )corresponding to a limit of sensitivity of 0.01–0.05 femtomol per spot. Orientating hCD200R using capture antibodies, showed that DX147 reacts with an epitope spatially distinct from the more closely related DX136 and OX108 epitopes. A panel of soluble recombinant proteins with mutations in hCD200R domain 1 produced by transiently transfected cells, was arrayed directly without purification and screened for binding to the three mAb. Several showed decreased binding to the blocking mAb DX136 and OX108, suggesting close proximity of these epitopes to the CD200 binding site. Binding of hCD200 to directly immobilized rat, mouse, and hCD200R was achieved with multimeric ligands, in the form of biotinylated-hCD200 coupled to FITC-labelled avidin coated beads. CONCLUSION: We have achieved sensitive, specific and reproducible detection of immobilized CD200R with different antibodies and mapped antigenic epitopes for two mAb in the vicinity of the ligand binding site using protein microarrays. We also detected CD200 binding to its receptor, a low affinity interaction, using beads presenting multivalent ligands. Our results demonstrate the quantitative aspects of protein arrays and their potential use in detecting simultaneously multiple protein-protein interactions and in particular the weak interactions found between leukocyte membrane proteins

OX133, a monoclonal antibody recognizing protein-bound N-ethylmaleimide for the identification of reduced disulfide bonds in proteins

In vivo, enzymatic reduction of some protein disulfide bonds, allosteric disulfide bonds, provides an important level of structural and functional regulation. The free cysteine residues generated can be labeled by maleimide reagents, including biotin derivatives, allowing the reduced protein to be detected or purified. During the screening of monoclonal antibodies for those specific for the reduced forms of proteins, we isolated OX133, a unique antibody that recognizes polypeptide resident, N-ethylmaleimide (NEM)-modified cysteine residues in a sequence-independent manner. OX133 offers an alternative to biotin-maleimide reagents for labeling reduced/alkylated antigens and capturing reduced/alkylated proteins with the advantage that NEM-modified proteins are more easily detected in mass spectrometry, and may be more easily recovered than is the case following capture with biotin based reagents

Crystal structure of signal regulatory protein gamma (SIRPγ) in complex with an antibody Fab fragment

BACKGROUND Signal Regulatory Protein γ (SIRPγ) is a member of a closely related family of three cell surface receptors implicated in modulating immune/inflammatory responses. SIRPγ is expressed on T lymphocytes where it appears to be involved in the integrin-independent adhesion of lymphocytes to antigen-presenting cells. Here we describe the first full length structure of the extracellular region of human SIRPγ. RESULTS We obtained crystals of SIRPγ by making a complex of the protein with the Fab fragment of the anti-SIRP antibody, OX117, which also binds to SIRPα and SIRPβ. We show that the epitope for FabOX117 is formed at the interface of the first and second domains of SIRPγ and comprises residues which are conserved between all three SIRPs. The FabOX117 binding site is distinct from the region in domain 1 which interacts with CD47, the physiological ligand for both SIRPγ and SIRPα but not SIRPβ. Comparison of the three domain structures of SIRPγ and SIRPα showed that these receptors can adopt different overall conformations due to the flexibility of the linker between the first two domains. SIRPγ in complex with FabOX117 forms a dimer in the crystal. Binding to the Fab fixes the position of domain 1 relative to domains 2/3 exposing a surface which favours formation of a homotypic dimer. However, the interaction appears to be relatively weak since only monomers of SIRPγ were observed in sedimentation velocity analytical ultracentrifugation of the protein alone. Studies of complex formation by equilibrium ultracentrifugation showed that only a 1:1 complex of SIRPγ: FabOX117 was formed with a dissociation constant in the low micromolar range (Kd = 1.2 +/- 0.3 μM). CONCLUSION The three-domain extracellular regions of SIRPs are structurally conserved but show conformational flexibility in the disposition of the amino terminal ligand-binding Ig domain relative to the two membrane proximal Ig domains. Binding of a cross-reactive anti-SIRP Fab fragment to SIRPγ stabilises a conformation that favours SIRP dimer formation in the crystal structure, though this interaction does not appear sufficiently stable to be observed in solution

Lack of effect of lowering LDL cholesterol on cancer: meta-analysis of individual data from 175,000 people in 27 randomised trials of statin therapy

<p>Background: Statin therapy reduces the risk of occlusive vascular events, but uncertainty remains about potential effects on cancer. We sought to provide a detailed assessment of any effects on cancer of lowering LDL cholesterol (LDL-C) with a statin using individual patient records from 175,000 patients in 27 large-scale statin trials.</p> <p>Methods and Findings: Individual records of 134,537 participants in 22 randomised trials of statin versus control (median duration 4.8 years) and 39,612 participants in 5 trials of more intensive versus less intensive statin therapy (median duration 5.1 years) were obtained. Reducing LDL-C with a statin for about 5 years had no effect on newly diagnosed cancer or on death from such cancers in either the trials of statin versus control (cancer incidence: 3755 [1.4% per year [py]] versus 3738 [1.4% py], RR 1.00 [95% CI 0.96-1.05]; cancer mortality: 1365 [0.5% py] versus 1358 [0.5% py], RR 1.00 [95% CI 0.93–1.08]) or in the trials of more versus less statin (cancer incidence: 1466 [1.6% py] vs 1472 [1.6% py], RR 1.00 [95% CI 0.93–1.07]; cancer mortality: 447 [0.5% py] versus 481 [0.5% py], RR 0.93 [95% CI 0.82–1.06]). Moreover, there was no evidence of any effect of reducing LDL-C with statin therapy on cancer incidence or mortality at any of 23 individual categories of sites, with increasing years of treatment, for any individual statin, or in any given subgroup. In particular, among individuals with low baseline LDL-C (<2 mmol/L), there was no evidence that further LDL-C reduction (from about 1.7 to 1.3 mmol/L) increased cancer risk (381 [1.6% py] versus 408 [1.7% py]; RR 0.92 [99% CI 0.76–1.10]).</p> <p>Conclusions: In 27 randomised trials, a median of five years of statin therapy had no effect on the incidence of, or mortality from, any type of cancer (or the aggregate of all cancer).</p&gt

Crop Updates 2002 - Cereals

This session covers thirty one papers from different authors: VARIETIES AND BREEDING 1. Agronomic evaluation of wheat and barley in the central wheatbelt of Western Australia, Peter Burgess1and Gary Fawell2, 1Agritech and 2Farmanco Management 2. Evaluating stress tolerance to terminal drought by Western Australian wheats, Dean Diepeveen and Dr Tim Setter, Department of Agriculture 3. Broadscale wheat variety comparisons featuring Wyalkatchem, Jeff Russell, Department of Agriculture 4. Australian crop accreditation system variety selector, Tony Seymour, Australian Crop Accreditation System 5. Future wheat varieties, Robin Wilson, Iain Barclay,Robyn McLean, Robert Loughman, Jenny Garlinge, Bill Lambe, Neil Venn and Peter Clarke, Department of Agriculture AGRONOMY 6. Beware of wheat variety interactions with row spacing and seed rate, Mohammad Amjad and Wal Anderson, Department of Agriculture 7. Yield and falling numbers of wheat varieties on the South Coast, Mohammad Amjad and Wal Anderson, Department of Agriculture 8. Maximising wheat variety performance through agronomic management, Wal Anderson, Raffaele Del Cima, James Bee, Darshan Sharma, Sheena Lyon, Melaine Kupsch, Mohammad Amjad, Pam Burgess, Veronika Reck, Brenda Shackley, Ray Tugwell, BindiWebb and Steve Penny Jr, Department of Agriculture 9. High impact of soil type and seasonal rainfall on optimum wheat seed rate , Raffaele Del Cima and Wal Anderson Department of Agriculture 10. 101 seasons in one day: Using the ‘WA Wheat’ database to predict wheat yield, James Fisher1, Bill Bowden1, Craig Scanlan1, Senthold Asseng2and Michael Robertson2 1Department of Agriculture, 2CSIRO 11. Economics of improving compact soils, M.A. Hamza1, G. McConnell2and W.K. Anderson1, 1Department of Agriculture, 2Planfarm 12. Reducing the risks in producing durum wheat in Western Australia, Md Shahajahan Miyan and Wal Anderson, Department of Agriculture 13. Taking the Why out of Wyalkatchem – the new widely adopted wheat variety, Steve Penny, Department of Agriculture 14. Influence of nutrition and environmental factors on seed vigour in wheat, Darshan Sharma, Wal Anderson and Daya Patabendige, Department of Agriculture NUTRITION 15. N and K are important for oat yield and quality, Patrick Gethin, Stephen Loss, Tim O’Dea, Ryan Guthrie and Lisa Leaver, CSBP Futurefarm 16. Effects of nitrogen and phosphorus on the grain yield and quality of noodle wheat, Tyrone Henning1, Lionel Martin1and Wal Anderson2 1Muresk Institute of Agriculture, 2Department of Agriculture 17. Assessment of a high input fertiliser regime on the yield and quality of Gairdner barley, Narelle Hill1, Simon Wallwork2and Laurence Carslake2 1Department of Agriculture, 2Wesfarmers Landmark 18. The use of Flexi-N to achieve high yielding, high protein wheat, Darren Hughes1, Lionel Martin1, Wal Anderson2and Stephen Loss3 1Muresk Institute of Agriculture, 2Department of Agriculture, 3CSBP Futurefarm 19. Are liquid phosphorus fertilisers more efficient than solid fertilisers in Western Australia?Stephen Loss, Lisa Leaver, Ryan Guthrie, Patrick Gethin and Tim O’Dea, CSBP Futurefarm 20. Oats respond to phosphorus and potassium, Glenn McDonald, Department of Agriculture PESTS AND DISEASES 21. Cereal disease diagnostics and rust monitoring, Nichole Burges and Dominie Wright, Department of Agriculture 22. Distribution and incidence of aphids and barley yellow dwarf virus in over-summering grasses in the Western Australian wheatbelt, Jenny Hawkes and Roger Jones, Centre for Legumes in Mediterranean Agriculture and Department of Agriculture 23. Spring sprays for powdery mildew control in cereals, Kith Jayasena1, Kazue Tanaka1, Vanessa Johnson1, Robert Loughman1and Josh Jury2 1Department of Agriculture, 2Wesfarmers Landmark 24. Impact of root lesion nematodes on wheat and triticale in Western Australia, Sean Kelly and Shashi Sharma, Department of Agriculture 25. Cropping options for the management of root lesion nematodes in Western Australia, Sean Kelly, Shashi Sharma and Robert Loughman, Department of Agriculture 26. Cereal rust update 2002 – new stem rust on Camm wheat, Robert Loughman1and Robert Park2 1Department of Agriculture, 2University of Sydney 27. Cereal aphids and direct feeding damage to cereals, Phil Michael, Department of Agriculture 28. A decision support system for control of aphids and BYDV in cereal crops, Debbie Thackray, Jenny Hawkes and Roger Jones, Department of Agriculture and Centre for Legumes in Mediterranean Agriculture STORAGE 29. Aeration – opportunity for profit, Christopher Newman, Department of Agriculture CLIMATE 30. Financial impact of frost on the Western Australian grains industry, Garren Knell and Kim Povey, ConsultAg 31. Summary of 2001 weather and seasonal prospects for 2002, David Stephens, Department of Agricultur