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.     

Sequences of cDNAs for human salivary and pancreatic α-amylases

The nucleotide sequences of the cloned human salivary and pancreatic α-amylase cDNAs correspond to the continuous mRNA sequences of 1768 and 1566 nucleotides, respectively. These include all of the amino acid coding regions. Salivary cDNA contains 200 bp in the 5′-noncoding region and 32 in the 3′-noncoding region. Pancreatic cDNA contains 3 and 27 bp of 5′- and 3′-noncoding regions, respectively. The nucleotide sequence humology of the two cDNAs is 96% in the coding region, and the predicted amino acid sequences are 94% homologous.Comparison of the sequences of human α-amylase cDNAs with those previously obtained for mouse α-amylase genes (Hagenbuchle et al., 1980; Schibler et al., 1982) showed the possibility of gene conversion between the two genes of human α-amylase.     

Abortive Infection of L Cells by Influenza B Virus: Defect in Bud Formation

Host-dependent restriction of influenza B virus replication in L cells was analysed in comparison with productive infection in MDCK or 1–5C-4 cells. The synthesis and intracellular distribution of virus-specific proteins and the production of cytoplasmic ribonucleoproteins in nonpermissive L cells were similar to those in permissive MDCK cells. However, an electron microscopic study of infected L cells showed neither extracellular virions nor budding virus particles on the cell surface, in contrast to MDCK cells which produced numerous virus particles. PAGE analysis of the plasma membrane isolated from the cells demonstrated no significant difference in the composition of viral polypeptides between permissive 1-5C-4 and nonpermissive L cells. It was noted that the abortiveness of influenza B virus infection in L cells may be due to a defect in host cell function involved in the initiation of virus budding.     

Plant-growth regulators from common starfish (<Emphasis Type="Italic">Asterias amurensis</Emphasis> Lütken) waste

Starfish waste has been shown to be an effective compost material not only in the promotion of plant growth but also in terms of having insecticidal activity. In the present study, plant growth regulation by chemicals from starfish was examined. The aqueous fraction from a hot water extract of the starfish Asterias amurensis Lütken showed plant-growth activity, while the aqueous fraction from a methanol extract inhibited growth of Brassica campestris. The lipophilic fraction from the methanol extract also exhibited a plant growth-promoting effect. The active components from each extract were identified. Asterubine from the hot water extract promoted plant growth. A ceramide from the lipophilic fraction showed root growth promoting effect, and three glucocerebrosides had promotive effects on the entire plant. Asterosaponins were identified as the main growth inhibitors in the aqueous fraction of the methanol extract. These active compounds from starfish waste could be analyzed as potential plant growth regulators in agricultural applications in the future.     

Cd-Binding Complexes from the Root Tissues of Various Higher Plants Cultivated in Cd2+-Containing Medium

Stone parsley, soybean, sunflower, sweet potato, potato, andadlay cultivated in a Cd²⁺-containing medium had Cd-bindingcomplexes with molecular weights of about 4,000 in the roottissues. The complexes were similar to the complex previouslyfound in water hyacinth roots in their absorption and CD spectraand their amino acid compositions. The results indicate thewidespread existence of complexes similar to fission yeast Cd-BPlin roots of various plants. (Received June 30, 1986; Accepted December 18, 1986)     

Properties and fluctuations in vivo of rat liver antizyme inhibitor.

Antizyme inhibitor was highly purified from rat liver by using affinity chromatography. It has some structural resemblance to ornithine decarboxylase (ODC), as judged from Mr, immunoreactivity and reversible binding with antizyme. However, unlike hepatic amounts of ODC and ODC-antizyme complex, that of antizyme inhibitor did not show much fluctuation upon putrescine treatment, whereas it decreased as rapidly as ODC decay in the presence of cycloheximide. These results suggested that antizyme inhibitor is an independent regulatory protein rather than a derivative of ODC. Changes in hepatic amounts of antizyme inhibitor, antizyme and ODC upon feeding suggested that antizyme inhibitor may play a role in ODC regulation by trapping antizyme and thereby suppressing ODC degradation. A monoclonal antibody to rat liver antizyme inhibitor was obtained. This antibody was shown to be utilizable for a simple assay of antizyme-inhibitor activity in tissue extracts.     

Determination of guanine by high-performance liquid chromatography with electrochemical detection: application to DNA and RNA assays

A highly sensitive assay for guanine was developed using high-performance liquid chromatography with electrochemical detection (ECD). Guanine was susceptible to the electrochemical oxidation, and ECD response was proportional to the amount of guanine in the range 0.25-4 pmol of guanine. The ECD of guanine was applicable to the analysis of nucleic acids. DNA and RNA were hydrolyzed in 0.03 and 3 M HCl, respectively, and guanine liberated from the nucleic acids was separated on a reverse-phase column and determined by ECD. The method allowed detection of 0.2 ng of calf thymus DNA or tRNA. An application of the method is shown for DNA and RNA assays in trichloroacetic acid extracts of rat adrenal and liver.     

Involvement of prostaglandin-induced proteins in the inhibition of herpes simplex virus replication

Cyclopentenone prostaglandin (PG), delta7-PGA1 was found to induce several polypeptides in human embryonic fibroblast (HEF) cells which were noticed to be dose-related and appeared after 1 h of treatment with a peak at around 5 h and gradual disappearance after 12 h. PG-induced proteins were almost identical in terms of molecular weights with those induced by heat-shock at 42 degrees C. Regarding the mechanism of inhibition of herpes simplex virus (HSV) replication by PG in cell culture, dot blot hybridization has revealed that the level of immediate early (IE) mRNA of the virus was reduced after PG treatment with time dependence. And this delayed inhibitory effect of delta7-PGA1 on HSV was shown to be associated with the production and accumulation of the induced polypeptides.