Modified peptide nucleic acids are internalized in mouse macrophages RAW 264.7 and inhibit inducible nitric oxide synthase.

Overexpression of inducible nitric oxide synthase causes the production of high levels of nitric oxide, which, under pathological conditions, leads to immunosuppression and tissue damage. The results recently obtained using peptide nucleic acids, rather than traditional oligonucleotides as antigen and antisense molecules, prompted us to test their efficacy in the regulation of nitric oxide production, thereby overcoming the obstacle of cellular internalization. The cellular permeability of four inducible nitric oxide synthase antisense peptide nucleic acids of different lengths was evaluated. These peptide nucleic acids were covalently linked to a hydrophobic peptide moiety to increase internalization and to a tyrosine to allow selective 125I radiolabelling. Internalization experiments showed a 3-25-fold increase in the membrane permeability of the modified peptide nucleic acids with respect to controls. Inducible nitric oxide synthase inhibition experiments on intact stimulated macrophages RAW 264.7 after passive permeation of the two antisense peptide nucleic acids 3 and 4 demonstrated a significant decrease (43-44%) in protein enzymatic activity with respect to the controls. These data offer a basis for developing a good alternative to conventional drugs directed against inducible nitric oxide synthase overexpression.     

Use of Radiation Hybrid panels to map genetic loci

The mapping of genetic loci within organisms has been accelerated by the advent of Radiation Hybrid (RH) panels. These panels are available for humans and non-humans including mice, baboon, rat, and canine. This article contains a general protocol for the use of the Genebridge 4 whole genome RH panel to map a human locus. This protocol may also be adjusted to suit the other RH panels currently available.     

Cell cycle dependent alterations of chromatin structure in situ as revealed by the accessibility of the nuclear protein AF-2 to monoclonal antibodies.

We have recently described a novel nuclear antigen, AF-2, which is related to cell cycle dependent alterations of chromatin structure. We show by two parameter flow cytometry on a cell by cell basis that the antigen is accessible to specific monoclonal antibodies only in mitotic and postmitotic early G1-phase cells. The evaluation of nuclease susceptibility and AF-2 antigen accessibility reveals different subcompartments of the G1-phase of the cell cycle with distinct chromatin conformations. Digestion with DNase I seems to alter the chromatin structure according to concentration and this is reflected by an increase of the antigen accessibility. Chromatin in the more condensed early G1-phase is specifically digested by lower concentrations of the enzyme than chromatin in later stages of interphase. Chromatin from cells in the late-G1, S-, and G2-phases shows a higher relative resistance to DNase I and a reduced accessibility of the AF-2 antigen to monoclonal antibodies. Nuclease S1 has a similar effect on chromatin topology, as revealed by the reaction with anti-AF-2 antibodies, without digestion of detectable amounts of DNA. The antigen becomes available to the antibodies in almost all cells by digestion with high concentrations of DNase I or Nuclease S1.     

Chemo-enzymatic synthesis of the Galili epitope Gal(alpha)(1-->3)Galbeta(1-->4)GlcNAc on a homogeneously soluble PEG polymer by a multi-enzyme system.

The alpha-Gal trisaccharide Gal(alpha)(1-->3)Galbeta(1-->4)GlcNAc 11 was synthesized on a homogeneously soluble polymeric support (polyethylene glycol, PEG) by use of a multi-enzyme system consisting of beta-1,4-galactosyltransferase (EC 2.4.1.38), alpha-1,3-galactosyltransferase (EC 2.4.1.151), sucrose synthase (EC 2.4.1.13) and UDP-glucose-4-epimerase (EC 5.1.3.2). In addition workup was simplified by use of dia-ultrafiltration. Thus the advantages of classic chemistry/enzymology and solid-phase synthesis could be united in one. Subsequent hydrogenolytic cleavage afforded the free alpha-Gal trisaccharide.     

The organization and structure of nerve and muscle in the jellyfish Cyanea capillata (coelenterata; scyphozoa)

The perirhopalial tissue and swimming muscle of Cyanea were examined with light microscopical and electron microscopical techniques. The perirhopalial tissue is a thin, triangular septum found on the subumbrellar surface of the animal. It separates part of the gastric canal system from the surrounding seawater, and is bound on two sides by radial muscle bands and on the third, the shorter side, by a rhopalium and the margin of the bell. The ectoderm of the perirhopalial tissue is composed of large, somewhat cuboidal, vacuolated, myoepithelial cells. The muscle tails of these cells form a single layer of radial, smooth muscle. Neurons of the “giant fiber nerve net” (GFNN), which form an extensive net over the perirhopalial tissue, lie at the base of the vacuolated portion of the myoepithelial cells. These neurons are visible in living tissue. The morphology of individual GFNN neurons was examined following intracellular injection of the fluorescent dye Lucifer Yellow. The neurons are usually bipolar and free of branches. At the electron microscope level, one usually finds that the GFNN neurons contain large vacuoles. The other characteristic feature of these cells is that they form symmetrical, or nonpolarized, synapses; that is, synaptic vesicles are found on both sides of the synapse. The swimming muscle is striated and composed of myoepithelial cells. Each myoepithelial cell has several muscle tails, and those of adjacent cells are linked to gether by desmosomes. The endoderm of the perirhopalial tissue also was examined. This investigation of the organization and ultrastructure of the perirhopalial tissue and surrounding muscle was undertaken to provide essential background information for an ongoing physiological study of the GFNN neurons and their synapses.     

The evolution of primate communities and societies in Madagascar

During their 120 to 165 million years of isolation, the flora and fauna of Madagascar evolved, to a large extent, independently of the African mainland.¹ In contrast to other oceanic islands, Madagascar is large enough to house the major components of tropical ecosystems, allowing tests of evolutionary hypotheses on the level of complete communities. Taking lemurs, the primates of Madagascar, as an example, evolutionary hypotheses correctly predict the organization of their community structure with respect to ecological correlates. Lemur social systems and their morphological correlates, on the other hand, deviate in some respects from those of other primates. Apparently, lemur social systems are influenced by several selection pressures that are weak or rare in other primates. These include variable activity patterns and avoidance of infanticide. The interspecific variation in lemur social systems therefore offers a unique opportunity for a comprehensive study of the determinants of primate social systems.