Effects of dimethylsulfoxide on the ultrastructure of fixed cells

It has been reported that the use of dimethylsulfoxide (DMSO) as a solvent for fixatives enhances preservation of cellular ultrastructure. By contrast, we have shown that DMSO alters the ultrastructural integrity of glutaraldehyde fixed cells. The cell membrane, nuclear envelope, endoplasmic reticulum, ribosomes, microtubules and intracytoplasmic organelles are most susceptible to the action of DMSO. We hypothesize that DMSO exerts intracellular alterations via its interaction with remnant interfacial water in fixed cells. DMSO-induced alterations of these and related cellular components may result in the formation of artefactual structures and networks. Thus, it appears that DMSO containing glutaraldehyde neither accelerates fixation nor enhances stabilization of cellular ultrastructure. For these reasons, addition of DMSO to fixatives is not recommended.     

The N Terminus of Pro-endothelial Monocyte-activating Polypeptide II (EMAP II) Regulates Its Binding with the C Terminus,Arginyl-tRNA Synthetase,and Neurofilament Light Protein

Pro-endothelial monocyte-activating polypeptide II (EMAP II), one component of the multi-aminoacyl tRNA synthetase complex, plays multiple roles in physiological and pathological processes of protein translation, signal transduction, immunity, lung development, and tumor growth. Recent studies have determined that pro-EMAP II has an essential role in maintaining axon integrity in central and peripheral neural systems where deletion of the C terminus of pro-EMAP II has been reported in a consanguineous Israeli Bedouin kindred suffering from Pelizaeus-Merzbacher-like disease. We hypothesized that the N terminus of pro-EMAP II has an important role in the regulation of protein-protein interactions. Using a GFP reporter system, we defined a putative leucine zipper in the N terminus of human pro-EMAP II protein (amino acid residues 1–70) that can form specific strip-like punctate structures. Through GFP punctum analysis, we uncovered that the pro-EMAP II C terminus (amino acids 147–312) can repress GFP punctum formation. Pulldown assays confirmed that the binding between the pro-EMAP II N terminus and its C terminus is mediated by a putative leucine zipper. Furthermore, the pro-EMAP II 1–70 amino acid region was identified as the binding partner of arginyl-tRNA synthetase, a polypeptide of the multi-aminoacyl tRNA synthetase complex. We also determined that the punctate GFP pro-EMAP II 1–70 amino acid aggregate colocalizes and binds to the neurofilament light subunit protein that is associated with pathologic neurofilament network disorganization and degeneration of motor neurons. These findings indicate the structure and binding interaction of pro-EMAP II protein and suggest a role of this protein in pathological neurodegenerative diseases.     

Parallel analysis of c-Fos protein and interleukin-2 expression in hypothalamic cells under different influence

The objective of this work was to perform a parallel analysis of activation of the rat anterior hypothalamus cells as judged by c-Fos protein expression, and of the expression of interleukin-2 (IL-2) under different influences, i. e., mild stress (handling) and adaptation to it, and intranasal administration of saline and the peptides Vilon (Lys-Glu) and Epithalon (Ala-Glu-Asp-Gly). Changes in the counts of cells positive for c-Fos- and IL-2 proteins were studied in structures of the lateral (LHA) area, anterior (AHN), supraoptic (SO) and paraventricular (PVH) nuclei of Wistar rat hypothalamus. Quantity of the interleukin-2-positive and c-Fos-positive cells was calculated. The findings were: a negative correlation between the activation of cells and the amount of IL-2 in the cells in the hypothalamic structures under study, and the specific patterns of changes in the counts of cells positive for c-Fos and IL-2 under stress and adaptation to stress.     

Cloning and expression of the gene for the mycoplasma key division protein FtsZ in Escherichia coli]

Gene ftsZ responsible for division of bacterial cells was revealed in most prokaryote groups. A 520-bp fragment of the ftsZ gene was amplified on the template of A. laidlawii DNA using degenerate primers. This fragment was sequenced and served as a hybridization probe for cloning of the full-sized copy of the A. laidlawii ftsZ gene. The amplified fragment was cloned in a pGEX3X vector and expressed in E. coli cells. Polyclonal antibodies derived from the chimeric polypeptide containing a fragment of A. laidlawii FtsZ protein interacted only with the A. laidlawii protein with molecular mass of 40 kDa. Comparison of nucleotide sequences of the ftsZ-gene region of A. laidlawii and other bacterial species showed that they were highly homologous in A. laidlawii, E. coli, and Bac. subtilis, while low homology was revealed between the A. laidlawii sequence and those of the members of the genus Mycoplasma. Analysis of the ftsZ-gene nucleotide sequences is suggested as a means to study the evolutionary relatedness of prokaryotes.     

Localization of carbohydrates in thick and ultrathin LR white-embedded tissue sections oxidized by acetic anhydride in dimethyl sulfoxide

The applicability of acetic anhydride (AA) in dimethyl sulfoxide (DMSO) for the oxidation of polysaccharide and their subsequent visualization with thiocarbohydrazide (TCH) and silver proteinate (SP) was evaluated on LR White-embedded thick and ultrathin liver sections. The results of these studies indicated that AA-DMSO-TCH-SP reaction is chemically specific on LR White-embedded tissues and that it offers distinct advantages for the localization of minute glycogen aggregates.     

Genetic manipulations and examination of differentiation properties of rat induced pluripotent cells

Induced pluripotent cells have offered new exciting options in fundamental and applied studies, such as gene knockout and human disease modeling. They are also very promising for regenerative medicine. However, in order to develop this technology, it is necessary to have a proper animal model. An appropriate model is the rat, which has physiological characteristics that are closer to human ones than do mice. In this paper, we present methods of genetic modifications with rat iPS cells and their directed differentiation. These data will help for studies using the rat as an experimental model for human replacement therapy.