Neomycin induces high-affinity agonist binding of G-protein-coupled receptors

Neomycin, an inositol-phospholipid-binding aminoglycoside antibiotic, is known to interfere with signal transduction mechanisms involving phospholipase C as effector enzyme. In this study, we report that neomycin can also markedly influence agonist binding of G-protein-coupled receptors. In membranes of differentiated human leukemia cells (HL 60 cells), neomycin (0.1-10 mM) was found to induce high-affinity binding of the chemotactic tripeptide, N-formyl-methionylleucylphenylalanine (fMet-Leu-Phe), to its receptor sites in a manner similar to magnesium. Gentamycin and streptomycin, two other aminoglycoside antibiotics, were as potent and as effective as neomycin or magnesium in inducing high-affinity agonist receptor binding. Pretreatment of the cells with pertussis toxin reduced the effects of magnesium and neomycin on agonist receptor binding likewise. In contrast, magnesium but not neomycin largely enhanced the potency of guanine nucleotides, particularly of GTP and its analog, guanosine-5'-O-(3-thiotriphosphate), to reduce fMet-Leu-Phe receptor binding, while maximal inhibition of agonist receptor binding by guanine nucleotides was identical with magnesium and neomycin. Furthermore, neomycin could not replace magnesium in providing stimulation of HL 60 membrane high-affinity GTPase by fMet-Leu-Phe. In close agreement to these findings on the pertussis-toxin-sensitive Gi-protein-coupled formyl peptide receptors, neomycin in a manner similar to magnesium induced high-affinity agonist binding of Gs-protein-coupled beta-adrenoceptors. Similar to formyl peptide receptor binding, high-affinity binding of isoproterenol to beta-adrenoceptors in guinea pig lung membranes induced by magnesium and neomycin was inhibited by the GTP analog, guanosine-5'-O-(3-thiotriphosphate), to a similar maximal extent but with an about 100-fold higher potency in the presence of magnesium than in the presence of neomycin. The data presented thus indicate that neomycin and other aminoglycoside antibiotics can mimic the action of magnesium (or other divalent cations) in inducing high-affinity agonist binding of Gi- and Gs-protein-coupled receptors, but not in inducing subsequent G-protein activation by guanosine triphosphates. The data, furthermore, suggest that neomycin by this selective action will be a powerful tool to dissect the multiple sites of magnesium's action in the agonist receptor-G-protein interaction.     

Progress in the histomorphometry of the microcirculatory system

Following a short review of the limits set to the procedures applied so far to measure quantitative changes in wall tissue of microvessels, a new measuring method is presented. It detect morphological reactions of the microcirculatory system on the grounds of changes in the numerical density of selectively visualized microvessels and their classification according to the external diameter by means of the automatic microscopic image analysing system QUANTIMET. Influences of structurally based and/or postmortal changes of the lumen wideness on the measurement are excluded by the automatic subtraction of the lumen area.     

Translocation ofLactobacillus murinus from the gastrointestinal tract

Bacterial translocation is defined as the passage of viable bacteria from the gastrointestinal tract to the mesenteric lymph nodes and other extraintestinal sites. The translocation rate of a newly described species of indigenous bacteria,Lactobacillus murinus, was compared with the translocation rates of indigenousLactobacillus acidophilus and nonindigenousSalmonella enteritidis. Groups of germfree or antibiotic-decontaminated, specific pathogen-free mice were monoassociated with each of these bacterial strains and tested at various intervals for translocation to the mesenteric lymph nodes. The translocation rates of the various bacteria expressed in decreasing order as the numbers of translocating bacteria per gram mesenteric lymph node wereS. enteritidis, L. murinus, andL. acidophilus. The degree of histologic damage to the gastrointestinal mucosa after monoassociation with these strains followed the same pattern. Thus,L. murinus translocates from the GI tract at a surprisingly high rate for an indigenous bacterial strain, and its translocation appears to be associated with mucosal alterations.     

Ultrastructure of plant chromosomes by high-resolution scanning electron microscopy

A technique is described for using standard squash preparations of mitotic and meiotic chromosomes for both light microscopy and subsequent high-resolution scanning electron microscopy for investigation of the same specimen. Depending on the microscope and conditions of preparation, a resolution of a few nanometers is routinely possible. Tilting of the specimen provides a three-dimensional insight into chromosomal structures. Combination of material-dependent signals of backscattered electrons with the secondary electron image allows an unambiguous localization of surface markers.     

Amino acid identities in the three redox center-carrying polypeptides of cytochromebc 1/b 6 f complexes

The comparison of primary structures is extended to 22 cytochromesb orb ₆, 12 cytochromesc ₁ orf, and 8 Rieske FeS proteins. Conclusions are drawn as to their phylogenetic relationship as well as on conserved, functionally important amino acids and secondary structures. The results are in favor of two independent quinone binding sites at opposite surfaces of the membrane, topping one of the two hemes of cytochromeb each.     

Glyphosate Induces 3-Deoxy-d-arabino-Heptulosonate 7-Phosphate Synthase in Potato (Solanum tuberosum L.) Cells Grown in Suspension Culture

The activity of the first enzyme of the shikimate pathway, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase, varies during the growth cycle of Solanum tuberosum L. cv superior cells in suspension culture. Maximum specific enzyme activity was observed midway through the linear phase of growth. When mid-log phase cells are exposed to glyphosate, the specific activity of the enzyme increases severalfold within 24 hours. The glyphosate-induced increase in enzyme activity is due to an increase in the amount of enzyme as determined by immunoblotting. Glyphosate (up to 2 millimolar) has no effect on the enzyme activity in vitro. Dehydroquinate synthase, the second enzyme of the shikimate pathway, is not induced by glyphosate.     

Interaction of CD2 with its ligand, LFA-3, in human T cell proliferation

Recently, it has been demonstrated that lymphocyte function-associated Ag (LFA-3) is a natural ligand for CD2 and that this receptor-ligand interaction functions in cell-cell adhesion. In this report, we demonstrate that LFA-3 plays a role in T cell activation. L cells were transfected with human genomic DNA and sorted for expression of LFA-3. We demonstrate that LFA-3+ L cells, together with anti-CD3 mAb or with suboptimal doses of PHA, stimulate proliferation of human peripheral blood T cells. Furthermore, thymocyte proliferation was induced by LFA-3+ L cells and suboptimal doses of PHA. Proliferation was inhibited by mAb directed against either CD2 or LFA-3. Stimulation of thymocytes by the combination of PHA and LFA-3+ L cells resulted in the increased expression of the IL-2R, as well as of the surface Ag 4F2, transferrin receptor, and HLA-DR. These data support the conclusion that LFA-3 plays a role in CD2-dependent T cell activation. LFA-3 is widely distributed and is expressed on all APC and target cells. Thus, the ability of the CD2/LFA-3 interaction to costimulate with an anti-CD3 mAb suggests that the CD2/LFA-3 interaction may be involved not only in an Ag-independent alternate pathway of T cell activation but also in Ag-specific T cell activation.