Bacterial metabolism, cytokine mRNA transcription and viability of bovine alveolar macrophages infected with Mycobacterium bovis BCG or virulent M. bovis

The effect of amino acid peroxides, relatively stable products of irradiation of amino acid solutions, on erythrocyte components was studied. Interaction of proline, lysine, valine, and leucine peroxides (100-300 mu M) with erythrocyte membranes brought about a decrease of membrane protein -SH group content and of activities of (Na+, K+)-ATPase and Ca2+ -ATPase, and induced aggregation of membrane proteins, due mainly to the formation of interpeptide disulfides. Interaction of amino acid peroxides with hemoglobin brought about hemoglobin oxidation to methemoglobin. The effects of amino acid peroxides are similar to those of t-butyl hydroperoxide. These results indicate that peroxides of amino acid and proteins, which can also be formed under physiological conditions, may be mediators of the cellular action of reactive oxygen species.     

The phosphorylation state of phosducin determines its ability to block transducin subunit interactions and inhibit transducin binding to activated rhodopsin

Heterotrimeric GTP-binding proteins (G-proteins) serve many different signal transduction pathways. Phosducin, a 28-kDa phosphoprotein, is expressed in a variety of mammalian cell types and blocks activation of several classes of G-proteins. Phosphorylation of phosducin by cyclic AMP-dependent protein kinase prevents phosducin-mediated inhibition of G-protein GTPase activity (Bauer, P. H., Müller, S., Puzicha, M., Pippig, S., Obermaier, B., Helmreich, E. J. M., and Lohse, M. J. (1992) Nature 358, 73-76). In retinal rods, phosducin inhibits transducin (Gt) activation by binding its beta gamma subunits. While rod phosducin is phosphorylated in the dark and dephosphorylated after illumination (Lee, R.-H., Brown, B. M., and Lolley, R. N. (1984) Biochemistry 23, 1972-1977), the significance of these reactions is still unclear. The data presented here permit a more precise characterization of phosducin function and the consequences of its phosphorylation. Dephosphophosducin blocked binding of the Gt alpha 1 subunit to activated rhodopsin in the presence of stoichiometric amounts of Gt beta gamma, whereas phosphophosducin did not. Surprisingly, the binding affinity of phosphophosducin for Gt beta gamma was not significantly reduced compared with the binding affinity of dephosphophosducin. However, the association of phosducin with Gt beta gamma in a size exclusion column matrix was dependent on the phosphorylation state of phosducin. Moreover, the ability of phosducin to compete with Gt alpha for binding to Gt beta gamma was also dependent on the phosphorylation state of phosducin. No interaction was found between phosducin and Gt alpha. These data indicate that phosducin decreases rod responsiveness by binding to the beta gamma subunits of Gt and preventing their interaction with Gt alpha, thereby inhibiting Gt alpha activation by the activated receptor. Moreover, phosphorylation of phosducin blocks its ability to compete with Gt alpha for binding to Gt beta gamma.     

Functional expression of two glucose transporter isoforms from the parasitic protozoan Leishmania enriettii

The parasitic protozoan Leishmania enriettii contains a family of tandemly repeated genes, designated Pro-1, that encode proteins with significant sequence similarity to mammalian facilitative glucose transporters. Pro-1 mRNAs are expressed almost exclusively in the promastigote or insect stage of the parasite life cycle. The Pro-1 tandem repeat encodes two isoforms of the putative transporter, iso-1 and iso-2, which have identical predicted amino acid sequences except for their NH2-terminal hydrophilic domains. We have now expressed both iso-1 and iso-2 by microinjecting their RNAs into Xenopus oocytes and assaying these oocytes for transport of various radiolabeled ligands. Both iso-1 and iso-2 transport [3H]2-deoxy-D-glucose, confirming that each protein is a bona fide glucose transporter. Each isoform also transports fructose and, to a much lesser degree, mannose. Compounds which inhibit 2-deoxy-D-glucose transport in L. enriettii promastigotes also inhibit transport in the microinjected oocytes expressing each isoform, indicating that the substrate specificities and pharmacological properties of each isoform are similar to those measured for 2-deoxy-D-glucose transport in intact parasites. The Km for transport of 2-deoxyglucose in oocytes expressing iso-1 is similar to that for oocytes expressing iso-2. These results reveal that both transporter isoforms have closely related functional properties and that the difference in their structures may serve some other purpose such as differential subcellular localization.     

Fluorescein angiographic characteristics of macular holes before and after vitrectomy with transforming growth factor beta-2

We evaluated the fluorescein angiographic features of full-thickness macular holes before and after vitrectomy, fluid-gas exchange, and instillation of transforming growth factor beta-2 in 43 eyes in a masked fashion to evaluate the angiographic characteristics of macular holes preoperatively and the changes that occur with successful and unsuccessful closure of the macular hole. Hyperfluorescence was present in the base of the macular hole preoperatively in 34 of 43 eyes (79.1%), was questionable in eight of 43 eyes (18.6%), and was definitely absent in only one of 43 eyes (2.3%). The hyperfluorescence in the base of the macular hole disappeared in 19 of 20 eyes (95%) with successful closure of the macular hole (P < .00001) and appeared to be caused by blocked fluorescence at the site of the macular hole. The photographic features of eyes with unsuccessful closure of the macular hole changed little, except that the size of the cuff of neurosensory detachment around the hole increased and was associated with decreased postoperative visual acuity. These angiographic changes support the presence of a glial tissue plug bridging a small defect in the fovea of eyes with successful closure of a macular hole.