Depicting the Spatial Distribution of Proteins in Human Tumor Tissue Combining SELDI and MALDI Imaging and Immunohistochemistry

Carcinoma tissue consists of not only tumor cells but also fibroblasts, endothelial cells or vascular structures, and inflammatory cells forming the supportive tumor stroma. Therefore, the spatial distribution of proteins that promote growth and proliferation in these complex functional units is of high interest. Matrix-assisted laser desorption/ionization imaging mass spectrometry is a newly developed technique that generates spatially resolved profiles of protein signals directly from thin tissue sections. Surface-enhanced laser desorption/ionization mass spectrometry (MS)combined with tissue microdissection allows analysis of defined parts of the tissue with a higher sensitivity and a broader mass range. Nevertheless, both MS-based techniques have a limited spatial resolution. IHC is a technique that allows a resolution down to the subcellular level. However, the detection and measurement of a specific protein expression level is possible only by semiquantitative methods. Moreover, prior knowledge about the identity of the proteins of interest is necessary. In this study, we combined all three techniques to gain highest spatial resolution, sensitivity, and quantitative information. We used frozen tissue from head and neck tumors and chose two exemplary proteins (HNP1–3 and S100A8) to highlight the advantages and disadvantages of each technique. It could be shown that the combination of these three techniques results in congruent but also synergetic data. (J Histochem Cytochem 58:929–937, 2010)     

The effect of substance P (SP) and SP partial sequences on nerve fiber growth in tissue culture

Explants of the ganglion trigeminale (PNS) and of the telencephalon (CNS) from chick embryos were cultivated in MAXIMOW chambers in semisynthetic media in the presence of dipeptide fragments (Lys(Z)-Pro . HCl, Lys-Pro-2HBr, Arg-Pro-2HCl) and the heptapeptide (SP5-11) of substance P as well as the complete substance P (SP1-11). 1. Histological examination of the dipeptide-treated CNS explants indicates that the structure of outgrowth in vitro is changed. Fascicel were observed. A stimulation of nerve fibre extension did not take place. 2.1. In dipeptide-treated PNS cultures the index of areas covered by the explants increased. 2.2. The index of nerve fibre growth increased significantly. The stimulation was caused in multiplication of fibres. Only Lys(Z)-Pro . HCl presents a prolongation of neurites. 2.3. SP5-11 effects in no case the growth of nerve fibres. SP1-11 stimulated significantly the fibre regeneration. 3. The possible role of SP1-11 with different effects under in vitro conditions is discussed. Only the N-terminal dipeptides stimulate the growth of nerve fibres. The C-terminal SP5-11 is without effect. Finally it is stated that the best results in neuritic enlargement and neurogenesis can only be obtained by cultivation with SP1-11.     

Serum antibodies to central nervous system antigens: An analysis of their relation with different human neurologic disorders

In a previous paper we have presented a double ligand enzyme linked immunosorbent assay (ELISA) technique suitable for the detection of human antibodies to different brain antigens. In the present study, we have applied this technique to the analysis of 100 neurologically affected patients with regard to both a list of clinical parameters and the presence in their sera of nervous tissue specific antibodies, in an attempt to highlight the meaning of such antibodies in different neurologic disorder. We show that the presence of these antibodies cannot be used for elucidation of pathogenesis or for diagnostic purposes, but can be used as a prognostic index.     

Retroviral elements and their hosts: insertional mutagenesis in the mouse germ line

The inbred mouse is an invaluable model for human biology and disease. Nevertheless, when considering genetic mechanisms of variation and disease, it is important to appreciate the significant differences in the spectra of spontaneous mutations that distinguish these species. While insertions of transposable elements are responsible for only ~0.1% of de novo mutations in humans, the figure is 100-fold higher in the laboratory mouse. This striking difference is largely due to the ongoing activity of mouse endogenous retroviral elements. Here we briefly review mouse endogenous retroviruses (ERVs) and their influence on gene expression, analyze mechanisms of interaction between ERVs and the host cell, and summarize the variety of mutations caused by ERV insertions. The prevalence of mouse ERV activity indicates that the genome of the laboratory mouse is presently behind in the “arms race” against invasion.     

Glutamine synthetase induction in chick embryo retina monolayers

Induction of glutamine synthetase (GS) by cortisol has been shown to occur in monolayer cultures of cells obtained by enzymatic dissociation of retinas from 8- and 12-day-old chick embryos with papain (0.1%) or trypsin (0.25%). Although essentially sigle cells when plated, monolayers obtained by enzymatic dissociation show significant aggregation by 4–6 h. Monolayers prepared by mechanical dispersion (cells forced through successively smaller gage needles) are minimally inducible, perhaps owing to poor viability in such cultures. Storage at 4°C for 24 h prior to treatment with cortisol significantly elevated both basal GS activity and inducibility in whole (but not in monolayer) retina cultures.     

Prohormone Thiol Protease and Enkephalin Precursor Processing: Cleavage at Dibasic and Monobasic Sites

Production of active enkephalin peptides requires proteolytic processing of proenkephalin at dibasic Lys-Arg, Arg-Arg, and Lys-Lys sites, as well as cleavage at a monobasic arginine site. A novel “prohormone thiol protease” (PTP) has been demonstrated to be involved in enkephalin precursor processing. To find if PTP is capable of cleaving all the putative cleavage sites needed for proenkephalin processing, its ability to cleave the dibasic and the monobasic sites within the enkephalin-containing peptides, peptide E and BAM-22P (bovine adrenal medulla docosapeptide), was examined in this study. Cleavage products were separated by HPLC and subjected to microsequencing to determine their identity. PTP cleaved BAM-22P at the Lys-Arg site between the two basic residues. The Arg-Arg site of both peptide E and BAM-22P was cleaved at the NH₂-terminal side of the paired basic residues to generate [Met]-enkephalin. Furthermore, the monobasic arginine site was cleaved at its NH₂-terminal side by PTP. These findings, together with previous results showing PTP cleavage at the Lys-Lys site of peptide F, demonstrate that PTP possesses the necessary specificity for all the dibasic and monobasic cleavage sites required for proenkephalin processing. In addition, the unique specificity of PTP for cleavage at the NH₂-terminal side of arginine at dibasic or monobasic sites distinguishes it from many other putative prohormone processing enzymes, providing further evidence that PTP appears to be a novel prohormone processing enzyme.