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.     

Formation of spray-dried powder of S-adenosyl-L-methionine

S-Adenosyl-L -methionine (SAM) is an essential metabolite in all living organisms. In clinical research, SAM has also been suggested as a chemotherapeutic agent in various diseases. The main problem of SAM is its instability at high temperatures, at neutral and alkaline pH, and in the presence of humidity. SAM retention in spray-dried powder was determined under various conditions of spray-drying. The highest SAM retention was obtained when maltodextrin (dextrose equivalent, DE, of 25) was used as the carrier solid with the SAM feed liquid at pH 4.0. The water content in the powder had a significant effect on the stability of SAM. SAM powder with lower water content exhibited higher stability.     

Radial dose distribution for 18.3 MeV/n alpha beams in tissue-equivalent gas

Experimental measurements of the radial restricted linear energy transfer (LETr) for alpha beams of 18.3 MeV/n in tissue-equivalent gas were presented. The radial dose distribution for the alpha beam was deduced from the restricted LET measurements. A differential W value for the alpha particle in the tissue-equivalent gas was also deduced. The result for the differential W value was 29.0 +/- 0.9 eV/ion pair. The radial dose varied according to an inverse-square function with distance from the track center for radii larger than 0.026 micron. The maximum extension of the track, the penumbra radius, as 2.73 +/- 1.67 microns, which was less than predicted by calculations (7-9 microns).     

Quantitative analysis of development of mitochondrial ultrastructure in differentiating mouse hepatocytes during postnatal period

Between birth and 10 days of age, the volume density (volume/unit cytoplasmic volume) of the matrix, and the surface density (area/unit cytoplasmic volume) of the inner membrane and cristae increased in both periportal and perihepatic hepatocytes, and did not differ significantly between the cells of the two zones. After 10 days of age, however, the volume density of the matrix decreased in perihepatic cells and remained unchanged in periportal cells, and, therefore, it became greater in periportal cells than in perihepatic cells in 20-day-old and adult animals. The surface density of the inner membrane and cristae decreased in the cells of both zones. Further, the hepatocyte volume increased markedly, especially in perihepatic zones between 20 days of age and the adult. The results show that, in postnatally differentiating hepatocytes, mitochondria are likely to develop during early postnatal period, then the structural heterogeneity of mitochondria arises, and hepatocyte volume increases markedly during late postnatal period after weaning. Thus, the process of postnatal hepatocyte differentiation includes such several phases of development.     

Heterogeneity and tissue-specific expression of eukaryotic transcription factor S-II-related protein mRNA

Two new cDNA clones for S-II-related proteins were isolated from a mouse liver cDNA library. Analyses of these clones suggested that S-II proteins are a family of proteins with relatively conserved C-terminal regions but variable N-terminal regions, and that some members of this family are expressed in a tissue-specific manner.     

Estimation of interaction function gamma(x) with sparsely ionizing radiation

The interaction function gamma(chi), which was introduced in the theory of dual radiation action as the probability that two energy transfers separated by distance chi combine with each other to produce a lesion, was estimated with sparsely ionizing radiation (60Co gamma rays and 40 kV X rays). Gamma(chi) was deduced on the assumption that the sensitive matrix is made up of small spherical flocculi distributed over the cell nucleus. The diameter of a flocculus was estimated at (4.0-11.2) X 10(-8) m when the diameter of the cell nucleus d was assumed to be 5 microns, and (4.0-11.4) X 10(-8) m when d was assumed to be 10 microns. It seems reasonable to hypothesize that the flocculus corresponds to the linker DNA in the chromatin structure of DNA, because the size of the linker DNA as a target (about 40 nm) is consistent with the diameter of flocculi obtained in this study.     

Analysis of a gene cluster for sarcotoxin II, a group of antibacterial proteins of Sarcophaga peregrina.

Sarcotoxin II is a group of antibacterial proteins of Sarcophaga peregrina (flesh fly) with related primary structures. We have cloned three genes in this family. These genes formed a tandem array with about 2-kb intervals, and one of them was present in the opposite strand. The putative amino acid sequences of the proteins encoded by these genes were very similar except for a deletion in one of them. All of the genes were found to be activated transiently in the same way when the larval body wall was injured, suggesting that the encoded proteins are acute-phase-responsive proteins for protecting the insect from bacterial infection.     

Glucagon receptors in endothelial and Kupffer cells of mouse liver

To determine whether hepatic sinusoidal cells contain glucagon receptors and, if so, to study the significance of the receptors in the cells, binding of [125I]-glucagon to nonparenchymal cells (mainly endothelial cells and Kupffer cells) isolated from mouse liver was examined by quantitative autoradiography and biochemical methods. Furthermore, the pathway of intracellular transport of colloidal gold-labeled glucagon (AuG) was examined in vivo. Autoradiographic and biochemical results demonstrated many glucagon receptors in both endothelial cells and Kupffer cells, and more receptors being present in endothelial cells than in Kupffer cells. In vivo, endothelial cells internalized AuG particles into coated vesicles via coated pits and transported the particles to endosomes, lysosomes, and abluminal plasma membrane. Therefore, receptor-mediated transcytosis of AuG occurs in endothelial cells. The number of particles present on the abluminal plasma membrane was constant if the amount of injected AuG increased. Therefore, the magnitude of receptor-mediated transcytosis of AuG appears to be regulated by endothelial cells. Kupffer cells internalized the ligand into cytoplasmic tubular structures via plasma membrane invaginations and transported the ligand exclusively to endosomes and lysosomes, suggesting that the ligand is degraded by Kupffer cells.     

(-) deprenyl induces activities of both superoxide dismutase and catalase but not of glutathione peroxidase in the striatum of young male rats

Daily s.c. injection of (-)deprenyl (2.0 mg/kg/day) for three weeks in young male rats caused a threefold increase in superoxide dismutase (SOD) activity in the striatum of the brain compared with the value in saline-injected control rats. Furthermore, the activity of catalase (but not of glutathione peroxidase) was also increased significantly by deprenyl treatment. The results confirmed the previous findings of Knoll on SOD activity and furthermore provided evidence that the activity of catalase is also significantly induced by the drug, which was not found in the previous study.     

Clerodane diterpenoids from the roots of Portulaca pilosa

Three trans-clerodane diterpenoids, pilosanol A, B and C, the last compound being a glucoside, have been isolated from the roots of Portulaca pilosa. They show a marked contrast in skeletal type with the constituents of aerial part. Evolutionary changes in the biosynthetic abilities of Portulaca species is discussed.