Chemical shift-based constraints for solution structure determination of paramagnetic low-spin heme proteins with bis-His and His-CN axial ligands: the cases of oxidized cytochrome b 5 and Met80Ala cyano-cytochrome c

The chemical shifts of the methyl protons of protoporphyrin IX, which are readily assigned, are related to the structural features of the axial histidine ligands in heme proteins with bis-His or His-CN axial coordination (Bertini I, Luchinat C, Parigi G, Walker FA (1999) JBIC 4:515-519). In the present paper, a module is developed which transforms the chemical shifts into a pseudo-potential energy that is a function of the dihedral angles defining the orientation of the axial ligand planes. Minimization of this pseudo-potential energy, together with the energetic contributions provided by the other constraints, yields structures consistent with the heme methyl chemical shifts. Oxidized cytochrome b(5) from rat and the cyanide derivative of the M80A mutant of yeast cytochrome c are used for test calculations. In the case of scarcity of NOEs for the axial ligands, owing to the presence of the paramagnetic center, the above structural constraints are shown to be quite precious. The newly refined structures are deposited in the PDB.     

Tumor-specific targeting of a cell line with natural killer cell activity by asialoglycoprotein receptor gene transfer

Targeting of immunological effector cells to tumor cells could be an efficient strategy of adoptive immunotherapy. The success of this strategy depends on the specificity of the effector cells and their availability in sufficient numbers. The aim of this study was to target the human natural killer cell line YT specifically to tumor cells. The cell line was modified by transfection with the cDNA of the human asialoglycoprotein receptor (ASGPR). This C-type lectin recognizes carbohydrates containing terminal galactosyl (Gal) residues, including the beta1-Gal bearing Thomsen-Friedenreich (TF) antigen, which is found on tumor cells. Binding assays revealed that the ASGPR-gene-transfected YT cell line binds significantly higher to tested target tumor cell lines than the mock-transfected control cells. Cytolytic activity against the tumor cell lines Raji, Jurkat and the TF-positive KG1 subline was increased. Genetic modification of YT cells could provide a useful tool for tumor targeting in immunotherapy.     

Small molecule inhibitors induce conformational changes in the I domain and the I-like domain of lymphocyte function-associated antigen-1. Molecular insights into integrin inhibition.

The beta(2) integrin lymphocyte function-associated antigen-1 (LFA-1) is a conformationally flexible alpha/beta heterodimeric receptor, which is expressed on the surface of all leukocytes. LFA-1 mediates cell adhesion crucial for normal immune and inflammatory responses. Intracellular signals or cations are required to convert LFA-1 from a nonligand binding to a ligand binding state. Here we investigated the effect of small molecule inhibitors on LFA-1 by monitoring the binding of monoclonal antibodies mapped to different receptor domains. The inhibitors were found to not only induce epitope changes in the I domain of the alpha(L) chain but also in the I-like domain of the beta(2) chain depending on the individual chemical structure of the inhibitor and its binding site. For the first time, we provide strong evidence that the I-like domain represents a target for allosteric LFA-1 inhibition similar to the well established regulatory L-site on the I domain of LFA-1. Moreover, the antibody binding patterns observed in the presence of the various inhibitors establish a conformational interaction between the LFA-1 I domain and the I-like domain in the native receptor that is formed upon activation. Differentially targeting the binding sites of the inhibitors, the L-site and the I-like domain, may open new avenues for highly specific therapeutic intervention in diseases where integrins play a pathophysiological role.     

Development of human and mouse strain of Hymenolepis nana in mice

Ferretti G., Gabriele F. and Palmas C. 1981. Development of human and mouse strain of Hymenolepis nana in mice. International Journal for Parasitology11: 425–430. An Hymenolepis nana strain isolated from human faeces was transferred in the mouse. At first, the q ratio (the number of parasites to the number of eggs used) was very low. After 10 passages adaptation was complete: when mice of different age and strain (CD₁ and BDF₁) were infected with eggs of the above strain and eggs of 2 different murine strains, no differences emerged in the q ratio or in the parasite mean dry weight. Survival also appeared the same in the 3 parasite strains, however, human and murine strains live for a shorter time in BDF₁ than in CD₁ mice. Discussion bears on the hypothesis that the difficulties that emerge during the first passages may be connected to the adult and not to the larval phase.     

Suitability of Macrolampis firefly and Pyrearinus click beetle luciferases for bacterial light off toxicity biosensor

Bioluminescence is widely used in biosensors. For water toxicity analysis, the naturally bioluminescent bacteria Vibrio fischeri have been used extensively. We investigated the suitability of two new beetle luciferases for Escherichia coli light off biosensors: Macrolampis firefly and Pyrearinus termitilluminans click beetle luciferases. The bioluminescence detection assay using this system is very sensitive, being comparable or superior to V. fischeri. The luciferase of P. termitilluminans produces a strong and sustained bioluminescence that is useful for less sensitive and inexpensive assays that require integration of the emission, whereas Macrolampis luciferase displays a flash-like luminescence that is useful for fast and more sensitive assays. The effect of heavy metals and sanitizing agents was analyzed. Zinc, copper, 1-propanol, and iodide had inhibitory effects on bioluminescence and growth assays; however, in these cases the bioluminescence was not a very reliable indicator of cell growth and metabolic activity because these agents also inhibited the luciferase. On the other hand, mercury and silver strongly affected cell bioluminescence and growth but not the luciferase activity, indicating that bioluminescence was a reliable indicator of cell growth and metabolic activity in this case. Finally, bioluminescent E. coli immobilized in agarose matrix gave a more stable format for environmental assays.