Mechanism of mda-5 Inhibition by Paramyxovirus V Proteins

The RNA helicases encoded by melanoma differentiation-associated gene 5 (mda-5) and retinoic acid-inducible gene I (RIG-I) detect foreign cytoplasmic RNA molecules generated during the course of a virus infection, and their activation leads to induction of type I interferon synthesis. Paramyxoviruses limit the amount of interferon produced by infected cells through the action of their V protein, which binds to and inhibits mda-5. Here we show that activation of both mda-5 and RIG-I by double-stranded RNA (dsRNA) leads to the formation of homo-oligomers through self-association of the helicase domains. We identify a region within the helicase domain of mda-5 that is targeted by all paramyxovirus V proteins and demonstrate that they inhibit activation of mda-5 by blocking dsRNA binding and consequent self-association. In addition to this commonly targeted domain, some paramyxovirus V proteins target additional regions of mda-5. In contrast, V proteins cannot bind to RIG-I and consequently have no effect on the ability of RIG-I to bind dsRNA or to form oligomers.     

Antigen-binding activity,structural characteristics and use of monoclonal antibodies to human alpha-1-microglobulin

Four monoclonal antibodies (mAbs), G6, F9, H8, and B2, against human alpha-1-microglobulin (A1M) have been produced and characterized. The parameters of affinity (K_p ～ 10⁹ M^?1), epitope specificity (the additively binding G6/F9, G6/H8, G6/B2, F9/H8, and F9/B2 pairs), and the observed effect of reversibility of structural changes induced by chemical agents allow use of these mAbs in biospecific methods of A1M purification and quantitative determination. The application of mAbs to an A1M enzyme immunoassay (analytical sensitivity—0.5 μg/l) and one step isolation of pure A1M by immunoaffinity chromatography was described.     

Saturation and Utilization of Nitrate Pools in Pea and Sugar Beet Leaves

The critical periods in the saturation of pea and sugar beet leaves with nitrate absorbed by roots were discriminated. In peas, during the first 14 h, all nitrate penetrating leaf cells was concentrated in the cytosol (metabolic pool). During the second period (14–62 h), nitrate began to flow into the vacuole (storage pool), and the filling of the metabolic pool continued. Metabolic pool was saturated by the end of this period (62 h). During the third period (62–110 h), further nitrate accumulation in the cell occurred because of expanding of the storage pool. Its saturation (similarly as total cell saturation) commenced 86 h after the start of nitrate uptake. In sugar beet leaves, both metabolic and storage nitrate pools were saturated by the end of the first period (14 h), and the sizes of these pools did not change during the second period (14–86 h). When pea plants were transferred to the nitrate-free medium, nitrate efflux began from the storage pool until its complete exhausting after 3 days. In sugar beet leaves, nitrate was still present in the storage pool 4 days after plant transfer to the nitrate-free medium. In both crops, nitrate export from the storage pool was aimed at the maintenance of the optimum nitrate concentration in the metabolic pool and, thus, at the maintenance of nitrate reductase activity. A functional diversity of nitrate compartmentation in the cells of various plant species is discussed.     

Aggresome formation by anti-Ras intracellular scFv fragments. The fate of the antigen-antibody complex.

Diverting the antigen from its normal intracellular location to other compartments in an antibody-mediated way represents a mode of action for intracellular antibodies [Cardinale, A., Lener, M., Messina, S., Cattaneo, A. & Biocca, S. (1998) FEBS Lett., 439, 197-202; Lener, M., Horn, I.R., Cardinale, A., Messina, S., Nielsen, U.B., Rybak, S.M., Hoogenboom, H.R., Cattaneo, A. & Biocca, S. (2000) Eur J Biochem. 267, 1196-205]. In the case of p21Ras, the sequestration of the antigen in aggregated structures in the cytoplasm of transfected cells leads to the inhibition of its biological function. We have further investigated the intracellular fate of the antigen-antibody complex by analyzing the effect of proteasome inhibitors on the formation and the intracellular localization of the aggregates. Overexpression of anti-Ras scFv fragments or inhibition of proteasomes activity leads to the formation of large perinuclear aggresomes formed of ubiquitinated-scFv fragments in which p21Ras is sequestered and degraded in an antibody-mediated way. Disruption of microtubules by nocodazole completely abrogates the accumulation of scFv fragments in a single aggresome and induces the dispersion of these structures in the periphery of the cell. Cotransfection of the GFP-scFv with a myc-tagged ubiquitin and colocalization with specific anti-proteasome antibodies indicate the recruitment of exogenous ubiquitin and proteasomes to the newly formed aggresomes. Taken together these results suggest that the intracellular antigen-antibody complex is naturally addressed to the ubiquitin-proteasome pathway and that the mechanism of ubiquitination does not inhibit the antibody binding properties and the capacity to block the antigen function.     

Hypoxic stress and the transport systems of the peribacteroid membrane of bean root nodules

When the roots of Vicia faba L. beans were subjected to hypoxic stress, the activity of H⁺-ATPase on the peribacteroid membrane, as well as the transport of dicarboxylates (malate and succinate) mediated by this enzyme, decreased. Since malate and succinate are the main carbon-containing metabolites involved in the energy supply to bacteroids, this caused a change of the relation type from mutualism to commensalism, and the domination of the eukaryotes over the prokaryotes consequently increased.