Protein aggregation and amyloid fibril formation by an SH3 domain probed by limited proteolysis

The SH3 domains are small protein modules of 60-85 amino acid residues that are found in many proteins involved in intracellular signal transduction. The SH3 domain of the p85alpha subunit of bovine phosphatidylinositol 3'-kinase (PI3-SH3) under acidic solution adopts a compact denatured state from which amyloid fibrils are readily formed. This aggregation process has been found to be modulated substantially by solution conditions. Here, we have analyzed the conformational features of the native and acid denatured states of PI3-SH3 by limited proteolysis experiments using proteinase K and pepsin, respectively. Moreover, we have analyzed the propensity of PI3-SH3 to be hydrolyzed by pepsin at different stages in the process of aggregation and amyloid formation at pH 1.2 and 2.0 and compared the sites of proteolysis under these conditions with the conformational features of both native and aggregated PI3-SH3. The results demonstrate that the denatured state of PI3-SH3 formed at low pH is relatively resistant to proteolysis, indicating that it is partially folded. The long loop connecting beta-strands b and c in the native protein is the region in this structure most susceptible to proteolysis. Remarkably, aggregates of PI3-SH3 that are formed initially from this denatured state in acid solution display enhanced susceptibility to proteolysis of the long loop, suggesting that the protein becomes more unfolded in the early stages of aggregation. By contrast, the more defined amyloid fibrils that are formed over longer periods of time are completely resistant to proteolysis. We suggest that the protein aggregates formed initially are relatively dynamic species that are able readily to reorganize their interactions to enable formation of very well ordered fibrillar structures. In addition, the disordered and non-native character of the polypeptide chains in the early aggregates could be important in determining the high cytotoxicity that has been revealed in previous studies of these species.     

Intra- and inter-laboratory variation in the scoring of micronuclei and nucleoplasmic bridges in binucleated human lymphocytes. Results of an international slide-scoring exercise by the HUMN project

One of the objectives of the HUman MicroNucleus (HUMN) project is to identify the methodological variables that have an important impact on micronucleus (MN) or micronucleated (MNed) cell frequencies measured in human lymphocytes using the cytokinesis-block micronucleus assay. In a previous study we had shown that the scoring criteria used were likely to be an important variable. To determine the extent of residual variation when laboratories scored cells from the same cultures using the same set of standard scoring criteria, an inter-laboratory slide-scoring exercise was performed among 34 laboratories from 21 countries with a total of 51 slide scorers involved. The results of this study show that even under these optimized conditions there is a great variation in the MN frequency or MNed cell frequency obtained by individual laboratories and scorers. All laboratories ranked correctly the MNed cell frequency in cells from cultures that were unirradiated, or exposed to 1 or 2Gy of gamma rays. The study also estimated that the intra-scorer median coefficient of variation for duplicate MNed cell frequency scores is 29% for unexposed cultures and 14 and 11% for cells exposed to 1 and 2Gy, respectively. These values can be used as a standard for quality or acceptability of data in future studies. Using a Poisson regression model it was estimated that radiation dose explained 67% of the variance, while staining method, cell sample, laboratory, and covariance explained 0.6, 0.3, 6.5, and 25.6% of the variance, respectively, leaving only 3.1% of the variance unexplained. As part of this exercise, nucleoplasmic bridges were also estimated by the laboratories; however, inexperience in the use of this biomarker of chromosome rearrangement was reflected in the much greater heterogeneity in the data and the unexplained variation estimated by the Poisson model. The results of these studies indicate clearly that even after standardizing culture and scoring conditions it will be necessary to calibrate scorers and laboratories if MN, MNed cell and nucleoplasmic bridge frequencies are to be reliably compared among laboratories and among populations.     

Selective interaction of ethidium derivatives with quadruplexes: an equilibrium dialysis and electrospray ionization mass spectrometry analysis

The telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structure, which has been shown to directly inhibit telomerase activity. The reactivation of this enzyme in immortalized and most cancer cells suggests that telomerase is a relevant target in oncology, and telomerase inhibitors have been proposed as new potential anticancer agents. In this paper, we have analyzed the selectivity of four ethidium derivatives and ethidium itself toward different G-quadruplex species, with electrospray mass spectrometry and competitive equilibrium dialysis and evaluated their inhibitory properties against telomerase. A selectivity profile may be obtained through electrospray ionization mass spectrometry (ESI-MS), which is in fair agreement with competitive equilibrium dialysis data. It also provides unambiguous data on the number of binding sites per nucleic acid (maximal number of two ethidium derivatives per quadruplex, in agreement with external stacking). Our experiments also demonstrate that one compound (4) is the most active and selective G-quadruplex ligand within this series and the most selective telomerase inhibitor in a modified TRAP-G4 assay.     

Increased acetylcholinesterase activities in specimens of Sparus auratus exposed to sublethal copper concentrations

The present study looks at possible changes in the activity of acetylcholinesterase (AChE) in tissues (brain and white muscle) of the Mediterranean bony fish Sparus auratus after a 20 days exposure to sublethal concentrations (0.1 or 0.5 ppm) of copper in the marine water and on control untreated animals. The trials also included measurements of Cu concentration in the tissues to evaluate possible metal accumulation. Moreover, sedimentation analysis as well as V(max) and K(m) determination were carried out in tissue extracts of Cu-exposed or control animals. V(max) and K(m) were also determined with or without addition of Cu(2+) in the assay. No Cu accumulation occurred in brain and muscle after Cu exposure. AChE showed in both tissues a molecular polymorphism with putative globular (G) and asymmetric (A) forms. Cu exposition led to an increased specific activity and improved catalytic efficiency of AChE in brain and muscle, seemingly regarding G forms. The increase in catalytic efficiency also resulted from the in vitro assay with tissue extracts and Cu(2+) addition. The higher AChE activity and catalytic efficiency in both tissues after Cu exposition and without metal accumulation, suggests an increase of free Cu aliquot into the cells, likely due to mechanisms of metal homeostasis.     

Lipid peroxidation in skeletal muscle of obese as compared to endurance-trained humans: a case of good vs. bad lipids?

Intra-myocellular triglycerides (IMTG) accumulate in the muscle of obese and endurance-trained (ET) humans and are considered a pathogenic factor in the development of insulin resistance, in the former. We postulate that this paradox may be associated with the peroxidation status of the IMTG. IMTG content was the same in the obese and ET subjects. The lipid peroxidation/IMTG ratio was 4.2-fold higher in the obese subjects. Hence, obesity results in an increased level of IMTG peroxidation while ET has a protective effect on IMTG peroxidation. This suggests a link between the lipid peroxidation/IMTG ratio and insulin resistance.     

Chromosome condensation defects in barren RNA-interfered Drosophila cells

Barren, the Drosophila homolog of XCAP-H, is one of three non-SMC subunits of condensin, a conserved 13S multiprotein complex required for chromosome condensation. Mutations in barren (barr) were originally shown to affect sister-chromatid separation during mitosis 16 of the Drosophila embryo, whereas condensation defects were not detected. In contrast, mutations in yeast homologs of barren result in defective mitotic chromosome condensation as well as irregular chromatid separation. We have used double-stranded RNA-mediated interference (RNAi) to deplete Barren in Drosophila S2 cells. Our analyses indicate that inactivation of barr leads to extensive chromosome condensation and disrupts chromatid segregation.     

Plasminogen activator production in a rat model of Pneumocystis carinii pneumonia

Several studies have indicated that the serine protease urokinase-plasminogen-activator (uPA) is an important factor in host defense against pulmonary pathogens. To gain a better insight into the role of uPA in Pneumocystis carinii (P. carinii) pneumonia (PCP), we evaluated PA production in alveolar macrophages (AMs) obtained from rats with steroid-induced PCP. Treatment with cortisone acetate favored PCP in 91% of rats. In the bronchoalveolar lavage (BAL) samples of immunosuppressed rats both with and without PCP, we observed a decrease in uPA activity as well as a decrease in cell number. Urokinase-PA production by AMs was reduced in rats treated with cortisone alone. However, an increase in cell-associated uPA was observed in rats with PCP. This increase appears to be produced in response to P carinii infection. In fact, when AMs obtained from untreated healthy or immunosuppressed uninfected rats were challenged with P carinii, a significant increase in PA activity in cell lysates was observed, though a lower response was obtained in cortisone-treated animals. Our results suggest that healthy AMs respond to the presence of P carinii with an increase in uPA production and that this response in immunodepressed rat-AMs is partially impaired.     

Bequest: The framework and directory of assessment methods

This paper has outlined the areas of the Environment and Climate Programme (Economic and Social Aspects of Human Settlement) the BEQUEST project addresses. It has also examined the framework for analysis the project sets out for a common understanding of SUD and the assessment methods currently made use of by planners, architects, engineers and surveyors to build environmental capacity. The paper has done this by: