An initial top-down proteomic analysis of the standard cuprizone mouse model of multiple sclerosis

An initial proteomic analysis of the cuprizone mouse model to characterise the breadth of toxicity by assessing cortex, skeletal muscle, spleen and peripheral blood mononuclear cells. Cuprizone treated vs. control mice for an initial characterisation. Select tissues from each group were pooled, analysed in triplicate using two-dimensional gel electrophoresis (2DE) and deep imaging and altered protein species identified using liquid chromatography tandem mass spectrometry (LC/MS/MS). Forty-three proteins were found to be uniquely detectable or undetectable in the cuprizone treatment group across the tissues analysed. Protein species identified in the cortex may potentially be linked to axonal damage in this model, and those in the spleen and peripheral blood mononuclear cells to the minimal peripheral immune cell infiltration into the central nervous system during cuprizone mediated demyelination. Primary oligodendrocytosis has been observed in type III lesions in multiple sclerosis. However, the underlying mechanisms are poorly understood. Cuprizone treatment results in oligodendrocyte apoptosis and secondary demyelination. This initial analysis identified proteins likely related to axonal damage; these may link primary oligodendrocytosis and secondary axonal damage. Furthermore, this appears to be the first study of the cuprizone model to also identify alterations in the proteomes of skeletal muscle, spleen and peripheral blood mononuclear cells. Notably, protein disulphide isomerase was not detected in the cuprizone cohort; its absence has been linked to reduced major histocompatibility class I assembly and reduced antigen presentation. Overall, the results suggest that, like experimental autoimmune encephalomyelitis, results from the standard cuprizone model should be carefully considered relative to clinical multiple sclerosis.     

Activity of redox-regulatory systems in the tumor and surrounding tissues in various histological types of tumors

According to modern concepts, a malignant tumor is a complex dynamic system possessing numerous links with both the immediate environment and remote non-malignant tissues and organs. Changes in their redox balance can result in disruption of the normal tissue control. The aim of our study was to compare activity of enzymes influencing the redox state in the tumor tissue, peritumoral area, and nonmalignant tissues (taken by resection line) at various histological tumor variants. We found similar close level of reduced glutathione in the tissues of gastric adenocarcinoma and vulvar squamous cell carcinoma; however, dynamics of this parameter in the tumor surrounding tissues was different. In contrast to gastric adenocarcinoma, vulvar squamous cell carcinoma was characterized by a significant increase in glutathione content in the tumor tissue and increased activity of all investigated enzymes of the glutathione system in the tumor tissue and its peritumoral area as compared with the surrounding non-malignant tissue. These results underlie existence of clear differences in the functioning of the redox regulatory systems in the tumor and surrounding tissues of various histological origin and localization; these differences may be possibly attributed to different mechanisms involved in maintenance of the redox balance in the originally non-malignant tissues.     

Conserved water-mediated H-bonding dynamics of catalytic Asn 175 in plant thiol protease

The role of invariant water molecules in the activity of plant cysteine protease is ubiquitous in nature. On analysing the 11 different Protein DataBank (PDB) structures of plant thiol proteases, the two invariant water molecules W1 and W2 (W220 and W222 in the template 1PPN structure) were observed to form H-bonds with the O_b atom of Asn 175. Extensive energy minimization and molecular dynamics simulation studies up to 2 ns on all the PDB and solvated structures clearly revealed the involvement of the H-bonding association of the two water molecules in fixing the orientation of the asparagine residue of the catalytic triad. From this study, it is suggested that H-bonding of the water molecule at the W1 invariant site better stabilizes the Asn residue at the active site of the catalytic triad.     

Na+ and K+ binding by glycerinated muscle fibers with reciprocal cation concentrations in the medium

The binding of Na+ and K+ by glycerinated muscle fibres was observed at reserve concentrations of NaCl in the medium. Under external concentrations of Na+ of K+ up to 0.4-0.5 mM, a constant fraction (0.15-0.25 mmoles/kg dry weight of the fibres) bound by glycerinated fibres was revealed. With the increase of NaCl or KCl concentration in the medium up to 10 mM the concentration of bound cations increased too. The parameters of Na+ and K+ sorption by glycerinated models were calculated. The values of Na+ and K+ binding limits were 4.4 and 1.8 mmole/kg dry weight of the fibres and those of affinity, 3.2 and 4.1 kcal/mol, respectively. The binding of one cation took place in conditions when its concentration was 10,000-20,000 fold less than that of the other cation. This points to the fact that Na+ and K+ binding is highly specific and is carried out by different centres. It is suggested that myosin ATPase is a substratum binding Na+ and K+ in glycerinated muscle fibres at reverse ratio concentrations of these cations in the medium.     

Amino acid sequence surrounding the lipoic acid cofactor of bovine kidney 2-oxoglutarate dehydrogenase complex

The 2-oxoglutarate dehydrogenase complex was succinylated using 2-oxo[5-14C]glutarate in the presence of N-ethylmaleimide to label the lipoic acid cofactor of the transuccinylase (E2) component. Following peptic digestion, 14C-lipoate-containing peptides were purified and subjected to automated Edman degradation and amino acid analysis. The amino acid sequence surrounding the lipoyllysine residue is reported.     

Early endosteal bone response to incorporated plutonium-238 in mice

Ten Swiss albino ICR SPF female mice 110 days old (weight about 30 g) were exposed for 48 hours to a solution of plutonium-238 nitrate (spec. act. 5 MBq/1 m1, pH 2.7) injected in amounts of 0.01 ml into the popliteal area of the right femur, each thus receiving about 500 kBq per 30 g body weight. Of the injected activity, 50% was retained in the right femur, 2% in the left femur and approximately 2-3% in the excrements collected separately from each animal during the whole exposure period. Ultrastructurally, electron micrographs revealed a variety of changes, including hypertrophy and destruction of endosteal cell organelles (primary damage), deformation and hypertrophy of osteocytes (secondary damage) and the irregularities in the osteocyte self-burial process leading to an abnormal formation of bone tissue structure (tertiary damage). Qualitatively, these changes in the irradiated bone ultrastructure were analogous to those occurring with age. This was confirmed by comparing two groups of control mice 110 and 330 days old. Assessed quantitatively, changes due to irradiation were more pronounced than those associated with aging.