Demonstration of a COOH-terminal extension on equine lutropin by means of a common acid-labile bond in equine lutropin and equine chorionic gonadotropin

The beta subunits of equine lutropin and equine chorionic gonadotropin were incubated in 0.013 N HCl for 30 min at 110 degrees C and separated into two fragments by reverse-phase high performance liquid chromatography. The amino acid and carbohydrate compositions of both fragments from each subunit were analyzed. The results demonstrated that equine lutropin-beta has a glycosylated COOH-terminal extension that differs only in carbohydrate composition from the COOH-terminal portion of equine chorionic gonadotropin-beta. This is the first demonstration of a glycosylated COOH-terminal extension in a pituitary glycoprotein hormone.     

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)     

A simple procedure to obtain one-micrometer sections of routinely embedded paraffin material

By freezing blocks of paraffin-embedded tissues to a convenient temperature it is possible to obtain routinely 1 micron sections that can be further processed as normal thicker sections. Normal and disposable steel knives can be used and the staining time should be increased in most procedures. Gradual freezing of blocks to the temperature of dry ice is the simplest and safest way to obtain an adequate temperature. The best results were obtained using as fixative 4% paraformaldehyde in phosphate buffered saline solution.     

Altered chromatin structure of cerebral nuclei in experimental diabetes mellitus.

To determine whether diabetes alters chromatin structure in vivo, micrococcal nuclease digestion kinetics were analyzed in cerebral cortical and hepatic nuclei of streptozotocin-induced diabetic rats. Cerebral nuclei of diabetic rats maintained for 6 weeks were less susceptible to micrococcal nuclease digestion compared with control rats. Insulin treatment reversed diabetes-related changes in nuclease digestion kinetics. There were no changes in the kinetics of digestion in hepatic nuclei. The reduced digestibility of cerebral DNA in diabetes could not be attributed to altered DNA fluorescence spectra, or altered distribution of most abundant chromatin proteins that were either solubilized or that remained insoluble immediately following nuclease digestion. It is concluded that chronic, uncontrolled hyperglycemia can alter chromatin structure of some tissues in vivo, and this change is probably related to subtle alterations in DNA-protein interactions.