Analysis of single Alzheimer solid plaque cores by laser capture microscopy and nanoelectrospray/tandem mass spectrometry

Aggregation of the 40-42 residue amyloid beta-peptide (Abeta) into amyloid plaques is a central event in Alzheimer's disease (AD) pathogenesis. Many proteins have by immunohistochemical techniques been shown to codeposit with Abeta in AD plaques. It is possible that some of these could seed Abeta aggregation and therefore be found in the actual core of the plaque. Here, we present a highly sensitive method for unbiased biochemical analysis of plaque cores. A mild purification protocol based on centrifugation and filtration was used to purify intact plaque cores from human AD brain. The purified plaques were dispensed on a glass slide and viewed in a laser capture microscope, and plaque cores were catapulted into a tube cap by a laser beam. After dissolution in formic acid, plaques were digested and analyzed by liquid chromatography coupled online to electrospray/tandem mass spectrometry. One single plaque was found to be sufficient for positive identification of the main amyloid component. Remarkably, Abeta was the only protein identified when 200 plaques were isolated and analyzed with the present method. Thus, it is possible that no proteins copolymerize with Abeta in the plaque cores and that Abeta alone is sufficient for formation of plaque cores. In support of this notion, core-like structures were observed after incubation of synthetic Abeta for 2 weeks. We suggest that the method described here could be used for the general analysis of amyloid aggregates and inclusion bodies found in other neurodegenerative disorders and that plaque cores in AD brain are molecularly homogeneous structures.     

Characterization of the endopeptidase activity of tripeptidyl-peptidase II

Tripeptidyl-peptidase II (TPP II) is a giant cytosolic peptidase with a proposed role in cellular protein degradation and protection against apoptosis. Beside its well-characterised exopeptidase activity, TPP II also has an endopeptidase activity. Little is known about this activity, and since it could be important for the physiological role of TPP II, we have investigated it in more detail. Two peptides, Nef(69-87) and LL37, were incubated with wild-type murine TPP II and variants thereof as well as TPP II from human and Drosophila melanogaster. Two intrinsically disordered proteins were also included in the study. We conclude that the endopeptidase activity is more promiscuous than previously reported. It is also clear that TPP II can attack longer disordered peptides up to 75 amino acid residues. Using a novel FRET substrate, the catalytic efficiency of the endopeptidase activity could be determined to be 5 orders of magnitude lower than for the exopeptidase activity.     

Proteomics and lipids of lipoproteins isolated at low salt concentrations in D2O/sucrose or in KBr

There is much interest in the significance of apolipoproteins and proteins that are noncovalently associated with lipoproteins. It is possible that the high ionic strength used for isolation of lipoproteins with KBr and NaI could alter the pattern of associated exchangeable proteins. Here we describe lipoprotein classes fractionation from up to 0.5 ml of serum or plasma with buffers of physiological ionic strength and pH prepared with deuterium oxide (D(2)O) and sucrose. An advantage of the D(2)O/sucrose procedure was that the lipoproteins could be directly analyzed by the techniques described without need for desalting. We compared the isolated lipoproteins with those obtained using ultracentrifugation in KBr from the same plasma pool. Electrophoretic homogeneity of the lipoproteins was very similar using the two methods, as well as their lipid composition evaluated by HPLC. Two-dimensional electrophoresis and surface-enhanced laser adsorption/ionization time-of-flight mass spectrometry indicated that the patterns of exchangeable proteins of VLDL isolated using with the two procedures were very similar. However, significant differences were found in the profiles of LDL and HDL, indicating that the D(2)O/sucrose method allowed a more complete characterization of its exchangeable apolipoproteins and proteins.     

Proteomic analysis of the cyanobacterium of the Azolla symbiosis: identity, adaptation, and NifH modification

Cyanobacteria are able to form stable nitrogen-fixing symbioseswith diverse eukaryotes. To extend our understanding of adaptationsimposed by plant hosts, two-dimensional gel electrophoresisand mass spectrometry (MS) were used for comparative proteinexpression profiling of a cyanobacterium (cyanobiont) dwellingin leaf cavities of the water-fern Azolla filiculoides. Homology-basedprotein identification using peptide mass fingerprinting [matrix-assistedlaser desorption ionization-time of flight (MALDI-TOF-MS)],tandem MS analyses, and sequence homology searches resultedin an identification success rate of 79% of proteins analysedin the unsequenced cyanobiont. Compared with a free-living strain,processes related to energy production, nitrogen and carbonmetabolism, and stress-related functions were up-regulated inthe cyanobiont while photosynthesis and metabolic turnover rateswere down-regulated, stressing a slow heterotrophic mode ofgrowth, as well as high heterocyst frequencies and nitrogen-fixingcapacities. The first molecular data set on the nature of theNifH post-translational modification in cyanobacteria was alsoobtained: peptide mass spectra of the protein demonstrated thepresence of a 300–400 Da protein modification localizedto a specific 13 amino acid sequence, within the part of theprotein that is ADP-ribosylated in other bacteria and closeto the active site of nitrogenase. Furthermore, the distributionof the highest scoring database hits for the identified proteinspoints to the possibility of using proteomic data in taxonomy. Key words: Azolla, cyanobacteria, NifH modification, proteomics, symbiosis, taxonomy Received 24 June 2007; Revised 21 October 2007 Accepted 22 October 2007     

Increased Salsolinol Levels in Rat Striatum and Limbic Forebrain Following Chronic Ethanol Treatment

Abstract: Endogenous levels of salsolinol and its methylated metabolite were measured by combined gas chromatography and mass spectrometry in rats chronically exposed to ethanol for 150 days. The chronic ethanol administration produced a significant increase of salsolinol concentrations in dopamine-rich brain areas, e.g., the striatum and the limbic forebrain. A negative correlation was observed between plasma ethanol concentration and the level of salsolinol in the brain. A possible role for salsolinol in the regulation of ethanol drinking and/or in the development of ethanol dependence is discussed.     

Influence of Thin Slice Reconstruction on CT Brain Perfusion Analysis

Objectives

Although CT scanners generally allow dynamic acquisition of thin slices (1 mm), thick slice (≥5 mm) reconstruction is commonly used for stroke imaging to reduce data, processing time, and noise level. Thin slice CT perfusion (CTP) reconstruction may suffer less from partial volume effects, and thus yield more accurate quantitative results with increased resolution. Before thin slice protocols are to be introduced clinically, it needs to be ensured that this does not affect overall CTP constancy. We studied the influence of thin slice reconstruction on average perfusion values by comparing it with standard thick slice reconstruction.

Materials and Methods

From 50 patient studies, absolute and relative hemisphere averaged estimates of cerebral blood volume (CBV), cerebral blood flow (CBF), mean transit time (MTT), and permeability-surface area product (PS) were analyzed using 0.8, 2.4, 4.8, and 9.6 mm slice reconstructions. Specifically, the influence of Gaussian and bilateral filtering, the arterial input function (AIF), and motion correction on the perfusion values was investigated.

Results

Bilateral filtering gave noise levels comparable to isotropic Gaussian filtering, with less partial volume effects. Absolute CBF, CBV and PS were 22%, 14% and 46% lower with 0.8 mm than with 4.8 mm slices. If the AIF and motion correction were based on thin slices prior to reconstruction of thicker slices, these differences reduced to 3%, 4% and 3%. The effect of slice thickness on relative values was very small.

Conclusions

This study shows that thin slice reconstruction for CTP with unaltered acquisition protocol gives relative perfusion values without clinically relevant bias. It does however affect absolute perfusion values, of which CBF and CBV are most sensitive. Partial volume effects in large arteries and veins lead to overestimation of these values. The effects of reconstruction slice thickness should be taken into account when absolute perfusion values are used for clinical decision making.     

Oak leaves as aerosol collectors: relationships with wind velocity and particle size distribution. Experimental results and their implications

Advancing the understanding of the aerosol-capture efficiencies of forest components such as leaves and needles, and of the mechanisms that underpin these efficiencies, is essential to the many related issues of forest turnover of nutrients and pollutants. For idealized collectors (such as artificial plates or cylinders) aerosol-mechanics offers a means for calculating capture efficiencies. For living collectors, in particular deciduous leaves, experimental investigations become necessary to assist in formulating the sub-models of capture efficiency that are fundamental to the modelling of fluxes of aerosol-borne substances to forests. We here present wind-tunnel based methods and results for leaves of Quercus robur L. exposed to an aerosol whose mass versus aerodynamic particle size distribution is characterised by a geometric mean aerodynamic particle diameter around 1.2 μm and a geometric standard deviation around 1.8. With respect to that distribution, and founded on a specially designed leaf wash-off method, we obtained average oak-leaf capture efficiencies ranging from 0.006% of the approaching aerosol mass flux at wind-speed 2 ms⁻¹ to 0.012% of the flux at wind-speeds 10 ms⁻¹, respectively. These values can be translated into deposition velocities (V _d ) for a leaf ensemble with a given leaf area index (LAI). With LAI in the range 2–5 (commonly found in the field) and for wind-speeds 2, 5 and 10 ms⁻¹, resulting V _d -values would be 0.02–0.05, 0.05–0.13, and 0.2–0.6 cm/s, respectively. To the extent comparisons are possible, our capture efficiency values are at the low end of the range of values reported by other researchers. The strong wind-speed sensitivity of V _d has implications for the deposition of aerosol-borne substances to forests for which wind regimes may shift as a result of climatic and land-use changes.