Evaluation of the symptomatic treatment of residual neurological symptoms in Wilson disease.

The intention of this analysis was to identify patients with treated Wilson disease (WD) and residual neurological symptoms in order to determine whether or not they were undergoing any treatment in addition to the common decoppering medication. Moreover, the effects of any symptomatic medication were analyzed. Two samples of WD patients were investigated either by a mailed questionnaire survey (n = 135) or by a retrospective analysis (n = 75). A considerable proportion of patients still suffered from neurological symptoms (n = 106, 50.5%), of whom a relatively small proportion was treated symptomatically (n = 33, 31.1%). The documented effects varied substantially, with anticholinergics and botulinum toxin (against dystonia) and primidone (against tremor) apparently being the most promising compounds. Further studies are required to analyze the symptomatic treatment of WD patients with residual neurological symptoms in more detail.

Localization of the actin-binding protein fesselin in chicken smooth muscle.

This report compares cellular localization of fesselin in chicken smooth, skeletal and cardiac muscle tissues using affinity purified polyclonal fesselin antibodies. Western blot analyses revealed large amounts of fesselin in gizzard smooth muscle with lower amounts in skeletal and cardiac muscle. In gizzard, fesselin was detected by immunofluorescence as discrete cytoplasmic structures. Fesselin did not co-localize with talin, vinculin or caveolin indicating that fesselin is not associated with dense plaques or caveolar regions of the cell membrane. Immunoelectron microscopy established localization of fesselin within dense bodies. Since dense bodies function as anchorage points for actin and desmin in smooth muscle cells, fesselin may be involved in establishing cytoskeletal structure in this tissue. In skeletal muscle, fesselin was associated with desmin in regularly spaced bands distributed along the length of muscle fibers suggesting localization to the Z-line. Infrequently, this banding pattern was observed in heart tissue as well. Localization at the Z-line of skeletal and cardiac muscle suggests a role in contraction of these tissues.

Improved MALDI-TOF imaging yields increased protein signals at high molecular mass.

Matrix assisted laser desorption ionization (MALDI) mass spectrum images are created from an array of mass spectra collected over a tissue surface. We have increased the mass range of proteins that can be detected in tissue sections from kidneys, heart, lung and brain of different rodent species by a modification of the sandwich technique, which involves co-crystallizing matrix with analyte. A tissue section is placed upon a drop of sinapinic acid matrix dissolved in 90% ethanol and 0.5% Triton X-100. Once the matrix has dried, a seed layer of sinapinic crystals is added as a dispersion in xylene. Additional layers of sinapinic acid are added as solutions in 90% ethanol followed by 50% acetonitrile. Numerous peaks with signal to noise ratio of four or greater are observed between 25 kDa to 50 kDa. This represents approximately 10 times as many peaks as are detected using traditional matrix spotting and spraying.

Evaluation of the Unified Wilson's Disease Rating Scale (UWDRS) in German patients with treated Wilson's disease.

Wilson's disease (WD) is an inherited autosomal-recessive disorder of copper metabolism characterized by a wide variety of neurological, hepatic, and psychiatric symptoms. The aim of the present study was the development and evaluation of a clinical rating scale, termed Unified Wilson's Disease Rating Scale (UWDRS), to assess the whole spectrum of clinical symptoms in WD. Altogether 107 patients (mean age 37.6 +/- 11.9 years; 46 male, 61 female) with treated WD participated in the study. Cronbach's alpha as a measure of the internal consistency for the entire scale was 0.92, whereas the intraclass correlation coefficient (ICC) was 0.98 (confidence interval (CI(95%)) 0.97-0.99), indicating an excellent interrater reliability as determined in 32 patients. Besides the total score was significantly correlated with the earning capacity of the patients as indicated by an estimated Spearman's rho approximately 0.54 (CI(95%) 0.40-0.69, P < 0.001). In summary, the UWDRS appears to be a promising tool to assess the disease severity in WD. Its usefulness in clinical research and drug trials should be further addressed.

Unified Wilson's Disease Rating Scale - a proposal for the neurological scoring of Wilson's disease patients.

BACKGROUND AND PURPOSE: The clinical forms of Wilson's disease (WD) neurological manifestations can be divided into three movement disorder syndromes: a) dystonic, b) ataxic, c) parkinsonian syndrome. These syndromes in WD seldom occur in isolation. Clinical rating scales such as the Unified Parkinson;s Disease Rating Scale (UPDRS), the International Cooperative Ataxia Rating Scale (ICARS) and the Rating Scale for Dystonia (RSD), focusing on either parkinsonism or ataxia or dystonia alone, are not sufficient to reflect accurately the motor impairment of WD patients. The aim of the study was to develop a novel rating scale for WD, because as far as we know no scale for the clinical rating in WD has been designed before. MATERIAL AND METHODS: In 2004 the EuroWilson consortium was founded, to create a European WD database. Members of the consortium from Poland, Germany, and France prepared a new scale using clinical rating scales as the UPDRS, ICARS, and RSD. Prepared drafts were discussed several times in detail at the first international neurological EuroWilson meeting in September 2004 in Paris and in November in Warsaw. RESULTS AND CONCLUSIONS: The novel scale for WD consists of 3 parts, including: consciousness, a historical review based on the Barthel scale (2-11 items), and neurological examination (12-35, items). The maximum score for the first part is 3, for the second 39 points, and for the last 143 points. The initial reliability of the scale on the basis of 6 patients (on DVD) and 8 investigators was assessed. Inter-rater agreement was high. Now the scale is used by the EuroWilson and GeNeMove consortia.

Detection of reversible protein thiol modifications in tissues.

Oxidation/reduction reactions of protein thiol groups (PSH) have been implicated in many physiological and pathological processes. Although many new techniques for separation and identification of modified cysteinyl residues in proteins have been developed, critical assessment of reagents and sample processing often are overlooked. We carefully compared the effectiveness of N-ethylmaleimide (NEM), iodoacetamide (IAM), and iodoacetic acid (IAA) in alkylating protein thiols and found that NEM required less reagent (125 vs. 1000 mol:mol excess), required less time (4 min vs. 4h), and was more effective at lower pHs (4.3 vs. 8.0) in comparison with IAM and IAA. The relative efficacy of dithiothreitol (DTT) and tris(2-carboxyethyl)phosphine (TCEP) for reducing protein disulfides suspended in NaPO(4) buffer or MeOH was assessed, and no differences in total normalized fluorescence were detected at the concentrations tested (10-100mM); however, individual band resolution appeared better in samples reduced with DTT in MeOH. In addition, we found that oxidation ex vivo was minimized in tissue samples that were homogenized in aqueous buffers containing excess molar quantities of NEM compared with samples homogenized in MeOH containing NEM. Using NEM for thiol alkylation, DTT for disulfide reduction, and mBBr for labeling the reduced disulfide and fluorimetric detection, we were able to generate an in-gel standard curve and quantitate total disulfide contents within biological samples as well as to identify changes in specific protein bands by scanning densitometry. We demonstrated that reagents and techniques we have identified for disulfide detection in complex samples are also applicable to two-dimensional electrophoresis separations.

Aggregation of ALS mutant superoxide dismutase expressed in Escherichia coli.

Although large amounts of wild-type human Cu,Zn superoxide dismutase (SOD) are easily expressed in Escherichia coli, the amyotrophic lateral sclerosis-associated mutants have a strong propensity to aggregate into inclusion bodies. The alanine to valine mutation at the fourth codon (A4V) is responsible for a rapidly progressive disease course and is particularly prone to aggregation when expressed in E. coli. We found that A4V SOD remained soluble when expressed at 18 degrees C, but >95% A4V SOD aggregated in inclusion bodies when expressed at 23 degrees C or above. The SOD aggregates dissolved with 4 M urea, suggesting that intermolecular hydrophobic interactions were predominantly responsible for making SOD insoluble. Many of the urea-solubilized subunits were cross-linked via disulfide bridges. Fully active mutant SOD could be produced by dialyzing urea away in the presence of beta-mercaptoethanol and subsequently adding copper plus zinc, providing a fast procedure for purifying hundreds of milligrams of protein. Extensive rinsing removed most contaminating E. coli proteins from A4V SOD inclusion bodies except for a 37 kDa protein identified as outer membrane protein F using MALDI ToF/ToF mass spectrometry. Our results indicate that metal-deficient ALS-mutant SOD folds into stable apo conformation able to rebind metals. At high protein concentrations, SOD forms aggregates through hydrophobic interactions between subunits that seem to act as a kinetic snare to entrap additional proteins.