Crystal structure of Y34F mutant human mitochondrial manganese superoxide dismutase and the functional role of tyrosine 34

Tyrosine 34 is a prominent and conserved residue in the active site of the manganese superoxide dismutases in organisms from bacteria to man. We have prepared the mutant containing the replacement Tyr 34 --> Phe (Y34F) in human manganese superoxide dismutase (hMnSOD) and crystallized it in two different crystal forms, orthorhombic and hexagonal. Crystal structures of hMnSOD Y34F have been solved to 1.9 A resolution in a hexagonal crystal form, denoted as Y34Fhex, and to 2.2 A resolution in an orthorhombic crystal form, denoted as Y34Fortho. Both crystal forms give structures that are closely superimposable with that of wild-type hMnSOD, with the phenyl rings of Tyr 34 in the wild type and Phe 34 in the mutant very similar in orientation. Therefore, in Y34F, a hydrogen-bonded relay that links the metal-bound hydroxyl to ordered solvent (Mn-OH to Gln 143 to Tyr 34 to H2O to His 30) is broken. Surprisingly, the loss of the Tyr 34 hydrogen bonds resulted in large increases in stability (measured by Tm), suggesting that the Tyr 34 hydroxyl does not play a role in stabilizing active-site architecture. The functional role of the side chain hydroxyl of Tyr 34 can be evaluated by comparison of the Y34F mutant with the wild-type hMnSOD. Both wild-type and Y34F had kcat/Km near 10(9) M-1 s-1, close to diffusion-controlled; however, Y34F showed kcat for maximal catalysis smaller by 10-fold than the wild type. In addition, the mutant Y34F was more susceptible to product inhibition by peroxide than the wild-type enzyme. This activity profile and the breaking of the hydrogen-bonding chain at the active site caused by the replacement Tyr 34 --> Phe suggest that Tyr 34 is a proton donor for O2* - reduction to H2O2 or is involved indirectly by orienting solvent or other residues for proton transfer. Up to 100 mM buffers in solution failed to enhance catalysis by either Y34F or the wild-type hMnSOD, suggesting that protonation from solution cannot enhance the release of the inhibiting bound peroxide ion, likely reflecting the enclosure of the active site by conserved residues as shown by the X-ray structures. The increased thermostability of the mutant Y34F and equal diffusion-controlled activity of Y34F and wild-type enzymes with normal superoxide levels suggest that evolutionary conservation of active-site residues in metalloenzymes reflects constraints from extreme rather than average cellular conditions. This new hypothesis that extreme rather than normal substrate concentrations are a powerful constraint on residue conservation may apply most strongly to enzyme defenses where the ability to meet extreme conditions directly affects cell survival.     

Clinical and functional investigation of 10 missense mutations and a novel frameshift insertion mutation of the gene for copper-zinc superoxide dismutase in UK families with amyotrophic lateral sclerosis

Mutations of the gene SOD-1, which encodes the enzyme copper-zinc superoxide dismutase, occur in patients with a familial form of amyotrophic lateral sclerosis (ALS). We investigated 71 families with more than one individual affected by ALS for clinical features and SOD-1 mutations. Mutations were identified in 14 families, indicating the presence of SOD-1 mutations in around 20% of this population. There were 10 different heterozygote missense point mutations in eight different codons, and a novel two-base frameshift insertion (132insTT), which leads to substitution of aspartic acid for glutamic acid at codon 132, and a premature stop codon at 133, with predicted truncation of the protein. SOD enzyme activity was reduced to around 50% of normal in individuals with SOD-1 mutations, and may be a useful predictor for the presence of these mutations. A predilection for disease onset in the lower limbs appears to be a distinguishing feature of familial ALS with SOD-1 mutations, and accords with findings in transgenic mouse models. In general, the finding of an SOD-1 mutation does not accurately predict a prognosis or disease severity.     

Release analysis and its use in the optimisation of the comminution and gravity circuits at the wheal jane tin concentrator

Use of Release Analysis on data obtained from laboratory testwork carried out on a Mozley Laboratory Mineral Separator is a powerful technique for optimising both gravity circuit performance and comminution requirements with respect to liberation size.This paper describes how the laboratory technique employed at Wheal Jane Mill has resulted in a significant improvement in the overall mill tin recovery.     

Selenocysteine's mechanism of incorporation and evolution revealed in cDNAs of three glutathione peroxidases

The nonsense codon, UGA, has for the first time recently beenshown to encode selenocysteine in two proteins, mouse glutathioneperoxidase (GSH-Px) (EC 1.11.1.9 [EC] ) and bacterial formate dehydrogenase.A co-translational rather than post-translational selenium-incorporationmechanism has been implicated. Furthermore, high expressionlevels of GSH-Px have suggested that suppression of terminationis efficient and specific. We have isolated and characterizedpituitary, kidney and placenta cDNAs for bovine, human and mouseGSH-Px respectively. It is demonstrated that this novel suppressionevent occurs in diverse tissues, in at least three mammalianspecies and at the translational step. Surprisingly, GSH-Pxis shown to be extramitochondrially encoded, indicating a cytosolicsuppression event rather than one utilizing the mitochondria'swell-documented extended codon-reading ability. Sequence analysisreveals that a simple proximal contextual pattern responsiblefor readthrough does not exist. Analysis of predicted secondarystrucutres of mRNAs, however, has revealed a conformation whichmay be unique to selenocysteine proteins and may prove usefulas a tool for artificial incorporation of selenocysteines. Ahuman intron for GSH-Px from an unspliced mRNA has been isolatedwhose position indicates an ancient, divergent evolutionaryrelationship with thioredoxin-S₂, rather than an independentconvergent one.     

A Versatile Evaporative Cooling System Designed for Use in an Elementary Particle Detector

An evaporative cooling system developed for operation and qualification testing of silicon pixel and microstrip detectors for the inner tracking detector of the CERN ATLAS spectrometer is described. Silicon detector substrates must be continuously operated between 0 and − 7°C in the high radiation environment near the circulating beams at the CERN Large Hadron Collider (LHC). This requirement imposes unusual constraints on the cooling system and has led to the choice of perfluoro-n-propane (C₃F₈) refrigerant, which combines good chemical stability under ionizing radiation with high dielectric strength and nonflammability. Since the silicon detectors must also be of extremely light construction to minimize undesirable physics background, coolant tubes are of thin (200 μm) aluminum wall, while evaporative operation allows a very low circulating coolant mass-flow (1–3 g · s⁻¹/100W to evacuate). The assembled detector arrays will undergo qualification tests at room temperature before installation in the ATLAS spectrometer. The cooling system is “dual-fuel,” and can also be operated with perfluoro-n-butane (C₄F₁₀) refrigerant, offering a reduced evaporation pressure (1.9 bar) compared to that of C₃F₈ (6.5 bar at 15°C). Paper presented at the Seventeenth European Conference on Thermophysical Properties, September 5–8, 2005, Bratislava, Slovak Republic.     

Human mitochondrial manganese superoxide dismutase polymorphic variant Ile58Thr reduces activity by destabilizing the tetrameric interface

Human manganese superoxide dismutase (MnSOD) is a homotetrameric enzyme which protects mitochondria against oxygen-mediated free radical damage. Within each subunit, both the N-terminal helical hairpin and C-terminal alpha/beta domains contribute ligands to the catalytic manganese site. Two identical four-helix bundles, symmetrically assembled from the N-terminal helical hairpins, form a novel tetrameric interface that stabilizes the active sites. The 2.5 A crystallographic structure of the naturally occurring polymorphic variant Ile58Thr MnSOD reveals that the helical hairpin mutation Thr58 causes two packing defects in each of the two four-helix bundles of the tetrameric interface. Similar mutations, expected to cause packing defects in the Cu,ZnSOD dimer interface, are associated with the degenerative disease amyotrophic lateral sclerosis. Ile58Thr MnSOD is primarily dimeric in solution and is significantly less thermostable than the normal enzyme, with decreases of 15 degrees C in the main melting temperature and 20 degrees C in the heat-inactivation temperature. Consequently, this mutant MnSOD is compromised at normal body temperatures: thermal inactivation, predicted from the decrease in thermal stability, occurs with a theoretical half-life of only 3.2 h at 37 degrees C (1.4 h at 41 degrees C), compared with 3.1 years for native MnSOD. This prediction is supported by direct measurements: incubation at 41.7 degrees C for 3 h has no effect on the activity of native MnSOD but completely inactivates mutant MnSOD. Rapid inactivation of Ile58Thr MnSOD at the elevated temperatures associated with fever and inflammation could provide an early advantage by killing infected cells, but also would increase superoxide-mediated oxidative damage and perhaps contribute to late-onset diseases.     

The closure of residential homes: what happens to residents

A controlled, prospective study is described which demonstrates changes in behaviour and mortality in the residents of two residential homes which were closed. The 59 residents were dispersed to 19 different establishments. Thirteen general practitioners (GPs) received sick elderly people on their lists after relocation. There were delays and other difficulties in communication between GPs. Restlessness, as measured by the Crichton Geriatric Behavioural Rating Scale, was increased soon after moving but no differences in behaviour or dependency between patients and control subjects were demonstrable after 1 year. Although moving in itself did not increase mortality, residents with poor mobility and dressing skills had a high death rate. This pattern of closure with widespread dispersal of residents is likely to become more common after the introduction of the NHS and Community Care Acts. It is associated with psychiatric morbidity which may be preventable by better organization. Medical involvement in the move was minimal, and the special responsibility of medical care of patients living in residential homes should be recognized.     

Why do few assistive technology systems make it to market? The case of the HandyHelper project

Assistive technologies, such as telecare monitoring applications installed in the home, are being promoted to help reduce pressure on health care systems caused by an aging population and as such promise a large market for new products. However, despite many projects undertaken by commercial companies, and despite significant investments both by the companies and by national and international funding programs in the EU, such systems are not widespread. This paper reports on a retrospective study of the development of one early system, HandyHelper. We were interested in what challenges the development team faced and why the system is no longer on the market. Qualitative research methods were applied, including document analysis and interviews of key people involved in its development. Even though the system worked technically, the input of older users was sought, and a pilot installation was run, the development was stopped. The findings from a thematic analysis point to complex issues. Some problems were avoidable, e.g., by providing more support for new users, though other problems point to inherent tensions, such as the different needs of sensor-based security features and interactive services aimed at the older users. Yet other aspects are outside of the developer’s control, such as available public funding. We summarize these findings and suggest lessons learnt for future projects.

     

Optimizing gaze control in three dimensions

Horizontal and vertical movements of the human eye bring new objects to the center of the visual field, but torsional movements rotate the visual world about its center. Ocular torsion stays near zero during head-fixed gaze shifts, and eye movements to visual targets are thought to be driven by purely horizontal and vertical commands. Here, analysis of eye-head gaze shifts revealed that gaze commands were three-dimensional, with a separate neural control system for torsion. Active torsion optimized gaze control as no two-dimensional system could have, stabilizing the retinal image as quickly as possible when it would otherwise have spun around the fixation point.