Quantitative proteome analysis in cardiovascular physiology and pathology. I. Data processing

Methodological evaluation of the proteomic analysis of cardiovascular-tissue material has been performed with a special emphasis on establishing examinations that allow reliable quantitative analysis of silver-stained readouts. Reliability, reproducibility, robustness and linearity were addressed and clarified. In addition, several types of normalization procedures were evaluated and new approaches are proposed. It has been found that the silver-stained readout offers a convenient approach for quantitation if a linear range for gel loading is defined. In addition, a broad range of a 10-fold input (loading 20-200 microg per gel) fulfills the linearity criteria, although at the lowest input (20 microg) a portion of protein species will remain undetected. The method is reliable and reproducible within a range of 65-200 microg input. The normalization procedure using the sum of all spot intensities from a silver-stained 2D pattern has been shown to be less reliable than other approaches, namely, normalization through median or through involvement of interquartile range. A special refinement of the normalization through virtual segmentation of pattern, and calculation of normalization factor for each stratum provides highly satisfactory results. The presented results not only provide evidence for the usefulness of silver-stained gels for quantitative evaluation, but they are directly applicable to the research endeavor of monitoring alterations in cardiovascular pathophysiology.

Integrated data acquisition and processing to determine metabolite contents, relaxation times, and macromolecule baseline in single examinations of individual subjects

Absolute quantitation of clinical (1)H-MR spectra is virtually always incomplete for single subjects because the separate determination of spectrum, baseline, and transverse and longitudinal relaxation times in single subjects is prohibitively long. Integrated Processing and Acquisition of Data (IPAD) based on a combined 2-dimensional experimental and fitting strategy is suggested to substantially improve the information content from a given measurement time. A series of localized saturation-recovery spectra was recorded and combined with 2-dimensional prior-knowledge fitting to simultaneously determine metabolite T(1) (from analysis of the saturation-recovery time course), metabolite T(2) (from lineshape analysis based on metabolite and water peak shapes), macromolecular baseline (based on T(1) differences and analysis of the saturation-recovery time course), and metabolite concentrations (using prior knowledge fitting and conventional procedures of absolute standardization). The procedure was tested on metabolite solutions and applied in 25 subjects (15-78 years old). Metabolite content was comparable to previously found values. Interindividual variation was larger than intraindividual variation in repeated spectra for metabolite content as well as for some relaxation times. Relaxation times were different for various metabolite groups. Parts of the interindividual variation could be explained by significant age dependence of relaxation times.

Digital Data Collection and Processing in Field and Lab: The Approach of Kinneret Regional Project

The paper showcases the field- and lab-documentation system developed for Kinneret Regional Project, an international archaeological expedition to the Northwestern shore of the Sea of Galilee (Israel) under the auspices of the University of Bern, the University of Helsinki, Leiden University and Wofford College. The core of the data management system is a fully relational, server-based database framework, which also includes time-based and static GIS services, stratigraphic analysis tools and fully indexed document/digital image archives. Data collection in the field is based on mobile, hand-held devices equipped with a custom-tailored stand-alone application. Comprehensive three-dimensional documentation of all finds and findings is achieved by means of total stations and/or high-precision GPS devices. All archaeological information retrieved in the field – including tachymetric data – is synched with the core system on the fly and thus immediately available for further processing in the field lab (within the local network) or for post-excavation analysis at remote institutions (via the WWW). Besides a short demonstration of the main functionalities, the paper also presents some of the key technologies used and illustrates usability aspects of the system’s individual components.

Visualization and quantification of air embolism structure by processing postmortem MSCT data.

Venous air embolism (VAE) is an often occurring forensic finding in cases of injury to the head and neck. Whenever found, it has to be appraised in its relation to the cause of death. While visualization and quantification is difficult at traditional autopsy, Magnetic Resonance Imaging (MRI) and Computed Tomography (CT) offer a new potential in the diagnosis of VAE. This paper reports the findings of VAE in four cases of massive head injury examined postmortem by Multislice Computed Tomography (MSCT) prior to autopsy. MSCT data of the thorax were processed using 3D air structure reconstruction software to visualize air embolism within the vascular system. Quantification of VAE was done by multiplying air containing areas on axial 2D images by their reconstruction intervals and then by summarizing the air volumes. Excellent 3D visualization of the air within the vascular system was obtained in all cases, and the intravascular gas volume was quantified.

Specific processing of tenascin-C by the metalloprotease meprinbeta neutralizes its inhibition of cell spreading

The metalloprotease meprin has been implicated in tissue remodelling due to its capability to degrade extracellular matrix components. Here, we investigated the susceptibility of tenascin-C to cleavage by meprinbeta and the functional properties of its proteolytic fragments. A set of monoclonal antibodies against chicken and human tenascin-C allowed the mapping of proteolytic fragments generated by meprinbeta. In chicken tenascin-C, meprinbeta processed all three major splicing variants by removal of 10kDa N-terminal and 38kDa C-terminal peptides, leaving a large central part of subunits intact. A similar cleavage pattern was found for large human tenascin-C variant where two N-terminal peptides (10 or 15kDa) and two C-terminal fragments (40 and 55kDa) were removed from the intact subunit. N-terminal sequencing revealed the exact amino acid positions of cleavage sites. In both chicken and human tenascin-C N-terminal cleavages occurred just before and/or after the heptad repeats involved in subunit oligomerization. In the human protein, an additional cleavage site was identified in the alternative fibronectin type III repeat D. Whereas all these sites are known to be attacked by several other proteases, a unique cleavage by meprinbeta was located to the 7th constant fibronectin type III repeat in both chicken and human tenascin-C, thereby removing the C-terminal domain involved in its anti-adhesive activity. In cell adhesion assays meprinbeta-digested human tenascin-C was not able to interfere with fibronectin-mediated cell spreading, confirming cleavage in the anti-adhesive domain. Whereas the expression of meprinbeta and tenascin-C does not overlap in normal colon tissue, inflamed lesions of the mucosa from patients with Crohn's disease exhibited many meprinbeta-positive leukocytes in regions where tenascin-C was strongly induced. Our data indicate that, at least under pathological conditions, meprinbeta might attack specific functional sites in tenascin-C that are important for its oligomerization and anti-adhesive activity.