Synthesis of 18O-labeled RNA for application to kinetic studies and imaging

Radioisotopes and fluorescent compounds are frequently used for RNA labeling but are unsuitable for clinical studies of RNA drugs because of the risk from radiation exposure or the nonequivalence arising from covalently attached fluorophores. Here, we report a practical phosphoramidite solid-phase synthesis of ¹⁸O-labeled RNA that avoids these disadvantages, and we demonstrate its application to quantification and imaging. The synthesis involves the introduction of a nonbridging ¹⁸O atom into the phosphate group during the oxidation step of the synthetic cycle by using ¹⁸O water as the oxygen donor. The ¹⁸O label in the RNA was stable at pH 3–8.5, while the physicochemical and biological properties of labeled and unlabeled short interfering RNA were indistinguishable by circular dichroism, melting temperature and RNA-interference activity. The ¹⁸O/¹⁶O ratio as measured by isotope ratio mass spectrometry increased linearly with the concentration of ¹⁸O-labeled RNA, and this technique was used to determine the blood concentration of ¹⁸O-labeled RNA after administration to mice. ¹⁸O-labeled RNA transfected into human A549 cells was visualized by isotope microscopy. The RNA was observed in foci in the cytoplasm around the nucleus, presumably corresponding to endosomes. These methodologies may be useful for kinetic and cellular-localization studies of RNA in basic and pharmaceutical studies.     

Production,Isolation and Pharmacological Studies of AI-77s

Two potent gastroprotective substances against experimental gastric ulcers in rats induced by stress and five of their analogues were isolated from the culture broth of bacterial strain AI-77 which was classified taxonomically as Bacillus pumilus. Physico-chemical properties and pharmacological activities of the seven compounds were examined and compared with each other.     

Electrophoretic Studies on Plant Protoplasts I. pH Dependence of Zeta Potentials of Protoplasts from Various Sources

Electrophoretic mobilities of plant protoplasts from varioussources were measured, as a function of the pH of the medium,by a micro-electrophoresis technique to characterize the protoplastsin terms of curves of zeta potential vs. protoplast surfacepH (pH_s). The shape of the curves of zeta potential vs. pH_scurves differed among preparations of protoplasts isolated fromvarious species and strains. The isoelectric points (pI) ofthe protoplasts measured in this study were between 3.0 and4.0. These differences among the protoplasts suggest that itmay be possible to develop an electrophoretic method for theselection of protoplasts. The shape of the curves of zeta potentialvs. pH_s also indicated that carboxyl groups, as well as phosphategroups, may contribute to the negative charges on the surfaceof protoplasts. (Received October 14, 1988; Accepted February 22, 1989)     

Enhancement of inositol phospholipid metabolism and activation of protein kinase C in ras-transformed rat fibroblasts

The inositol phospholipid metabolism is one of the main pathways of signal transduction in cells. We measured the activities of its key enzymes in v-Ha-ras-transformed 208F rat fibroblasts. In the ras-transformed clones, incorporation of [32P]Pi into intermediates of the inositol phospholipid metabolism was stimulated. The activities of phosphatidylinositol and phosphatidylinositol-4-phosphate kinases in the transformed clones were about 35-50% more than in untransformed cells, indicating increased inositol phospholipid metabolism. However, the activity of diacylglycerol kinase in their membrane fraction was 25-35% less than that of untransformed cells, although the total diacylglycerol kinase activity did not change. The imbalance of these kinases could constitute one of the main reasons leading to the increased level of inositol phosphates and the accumulation of diacylglycerol to 2-2.2 times that in control 208F cells. Phosphatidylinositol-4,5-bisphosphate-phospholipase C activity did not change on the transformation when assayed under various conditions. The increased level of diacylglycerol caused intracellular translocation, activation, and down-regulation of protein kinase C changes which may be one of the essential events in transformation by the v-Ha-ras gene.     

Production of monoclonal antibodies against Clostridium botulinum type E derivative toxin

Abstract Five monoclonal antibodies (MCA; E–8–2, 9–1, 11–2, 12–4, and 13–1) against Clostridium botulinum type E derivative toxin were prepared. Their ELISA titers were higher than or equivalent to that of conventional polyclonal antibody. Three of them (E-8–2, 12–4, and 13–1) possessed the neutralizing activity comparable to that of polyclonal antibody. The results of binding-competition experiments indicated that the monoclonal antibodies bound to different sites on the type E toxin molecule. Immunoblotting analyses demonstrated that E-8–2, 9–1, and 11–2 react to fragment I (heavy chain) of the toxin. By use of these monoclonal antibodies, it may be possible to scrutinize the structure-function relationship of botulinum toxins and cross reactions between type E and F toxins.     

Use of monoclonal antibodies in enzyme linked immunosorbent assay (ELISA) for detection of botulinum type B toxins

Use of polyclonal antibodies failed to correlate mouse assay with enzyme linked immunosorbent assay (ELISA) in titration of culture fluid of different strains of Clostridium botulinum type B. If ELISA is performed with such a monoclonal antibody that is capable of neutralizing the toxin, however, the lethal toxicity can be determined quantitatively.