Lipid fluidity and composition of the erythrocyte membrane from healthy dogs and labrador retrievers with hereditary muscular dystrophy

Erythrocyte membranes and their liposomes were prepared from clinically normal dogs and Labrador retrievers with hereditary muscular dystrophy. The static and dynamic components of fluidity of each membrane were then assessed by steady-state fluorescence polarization techniques using limiting hindered fluorescence anisotropy and order parameter values of 1,6-diphenyl-1,3,5-hexatriene (DPH) and fluorescence anisotropy values ofdl-2-(9-anthroyl)-stearic acid anddl-12-(9-anthroyl)-stearic acid, respectively. Membrane lipids were extracted and analyzed by thin-layer chromatography and gas chromatography. The results of these studies demonstrated that the lipid fluidity of erythrocyte membranes, and their liposomes, prepared from dystrophic dogs were found to possess significantly lower static and dynamic components of fluidity than control counterparts. Analysis of the composition of membranes from dystrophic dogs revealed a higher ratio of saturated fatty acyl chain/unsaturated chains (w/w) and lower double-bond index. Alterations in the fatty acid composition such as decrease in levels of linoleic (18:2) and arachidonic (20:4) acids and increase in palmitic (16:0) and stearic (18:0) acids were also observed in the membranes of dystrophic animals. These associated fatty acyl alterations could explain, at least in part, the differences in membrane fluidity between dystrophic and control dogs.     

Immunological analysis of histone H2A from the slime mold Physarum polycephalum

Specific antibodies against the histone H2A from calf thymus were generated by injecting rabbits with complexes: histone H2A-RNA with a protein to RNA ratio of 3:1. In the microcomplement fixation assay the antibodies against the histone H2A from calf thymus immuno-reacted with the histone H2A from calf thymus but not with H2A from Physarum polycephalum. The histone H2A from calf thymus therefore appears to have an immunological determinant(s) which does not exist in H2A from Physarum polycephalum.     

Distinct residues of human p53 implicated in binding to DNA, simian virus 40 large T antigen, 53BP1, and 53BP2.

We identified a minimal domain of human p53 required for the transactivation of a p53 response element in Saccharomyces cerevisiae. This domain contains the central region of p53 sufficient for specific DNA binding, which colocalizes with the region responsible for binding simian virus 40 large T antigen, 53BP1, and 53BP2. Thirty amino acid positions, including natural mutational hot spots (R175, R213, R248, R249, and R273), in the minimal DNA-binding domain were mutated by alanine substitution. Alanine substitutions at positions R213, R248, R249, D281, R282, R283, E286, and N288 affected transactivation but allowed binding to at least one of the three interacting proteins; these amino acids may be involved in amino acid-base pair contacts. Surprisingly, alanine substitution at the mutational hot spot R175 did not affect DNA binding, transactivation, or T-antigen binding, although it nearly eliminated binding to 53BP1 and 53BP2. Mutation of H168 significantly affected only T-antigen binding, and mutation of E285 affected only 53BP1 binding. Thus, we implicate specific residues of p53 in different DNA and protein interactions.     

Chromatin proteins associated with micrococcal nuclease-sensitive and nuclease-resistant chromatin fractions of Kirkman-Robbins hepatoma and hamster liver

Micrococcal nuclease-sensitive (SP) and nuclease-resistant (PP) chromatin fractions from Kirkman-Robbins hepatoma and hamster liver were obtained. The molecular distribution of three non-histone proteins (NHCP1, NHCP2 and NHCP3), histones, and chromatin-bound protease activity between SP and PP fractions of both tissues was compared. Differences, mainly of quantitative nature, among non-histone proteins of neoplastic and normal tissue were observed. Moreover, it was found that polypeptides with mol. wt 81 000 (NHCP1), 39 000 (NHCP2) and 21 000, 35 000, 37 000 (NHCP1), 70 000, 112 000, 141 000, 157 000 (NHCP2), 30 000–33 000 (NHCP3) were associated only with the nuclease-sensitive part of chromatin of hepatoma and normal tissue, respectively. A major difference in histone compostion of hamster hepatoma and liver concerns histones H2A and H1. Furthermore, an enrichment of high mobility group proteins as well as other soluble non-histone proteins in an acid extract of the SP fraction was observed. Apparently chromatin-bound protease activity can be found in both fractions of chromatin.     

Presynaptic Glutamate Receptors Regulate Noradrenaline Release from Isolated Nerve Terminals

The wide-ranging neuronal actions of excitatory amino acids, such as glutamate, are thought to be mediated mainly by postsynaptic N-methyl-D-aspartate (NMDA) and non-NMDA receptors. We now report the existence of presynaptic glutamate receptors in isolated nerve terminals (synaptosomes) prepared from hippocampus, olfactory bulb, and cerebral cortex. Activation of these receptors by NMDA or non-NMDA agonists, in a concentration-dependent manner, resulted in Ca(2+)-dependent release of noradrenaline from vesicular transmitter stores. The NMDA-stimulated release was potentiated by glycine and was blocked by Mg2+ and selective NMDA antagonists. In contrast, release stimulated by selective non-NMDA agonists was blocked by 6-cyano-7-nitroquinoxaline-2,3- dione, but not by Mg2+ or NMDA antagonists. Our data suggest that the presynaptic glutamate receptors can be classified pharmacologically as both the NMDA and non-NMDA types. These receptors, localized on nerve terminals of the locus ceruleus noradrenergic neurons, may play an important role in interactions between noradrenaline and glutamate.     

Structure of the sialic acid-containing O-specific polysaccharide from Salmonella enterica serovar Toucra O48 lipopolysaccharide.

Lipopolysaccharide was extracted from cells of Salmonella enterica serovar Toucra O48 and, after mild acid hydrolysis (1% AcOH, 1 h, 100 degrees C or 0.1 M NaOH-AcOH, pH 4.5, 5 h, 100 degrees C), the O-specific polysaccharide was isolated and characterized. The core and an oligosaccharide containing a fragment of the repeating unit linked to the core region were also obtained, depending on hydrolysis conditions. On the basis of sugar and methylation analyses and NMR spectroscopy of the hydrolysis products, the biological repeating unit of the O-specific polysaccharide was shown to be the following trisaccharide: -->4)-alpha-Neup5Ac(2-->3)-L-alpha-FucpNAc(1-->3)-D-beta-Glc pNAc(1--> The polysaccharide O-chain was substituted with a single molar equivalent of O-acetyl group, distributed between the Neu5Ac O-9 and O-7 positions, in an approximate ratio of 7 : 3.     

Transgenic pigs designed to express human α-galactosidase to avoid humoral xenograft rejection

The use of animals as a source of organs and tissues for xenotransplantation can overcome the growing shortage of human organ donors. However, the presence of xenoreactive antibodies in humans directed against swine Gal antigen present on the surface of xenograft donor cells leads to the complement activation and immediate xenograft rejection as a consequence of hyperacute reaction. To prevent hyperacute rejection, it is possible to change the swine genome by a human gene modifying the set of donor’s cell surface proteins. The gene construct pGal-GFPBsd containing the human gene encoding α-galactosidase enzyme under the promoter of EF-1α elongation factor ensuring systemic expression was introduced by microinjection into a male pronucleus of the fertilised porcine oocyte. As a result, the founder male pig was obtained with the transgene mapping to chromosome 11p12. The polymerase chain reaction (PCR) analysis revealed and the Southern analysis confirmed transgene integration estimating the approximate number of transgene copies as 16. Flow cytometry analysis revealed a reduction in the level of epitope Gal on the cell surface of cells isolated from F0 and F1 transgenic animals. The complement-mediated cytotoxicity assay showed increased viability of the transgenic cells in comparison with the wild-type, which confirmed the protective influence of α-galactosidase expression.