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.     

Analysis of water in Autonomous Biological Systems (ABS) samples.

Several soluble components, peptidase and amino acids, and carbon isotopic ratio in the water retrieved from flight experiments of Autonomous Biological Systems (ABS) as well as ground control samples are analyzed to interpret the condition, dynamics, material balance of the ABS ecosystems. Organic carbons in flight samples were found to be more abundant compared with the control ones, which suggested the uniform ecosystems in low gravity might easily dissolve more soluble components. The Mir-1997 flight sample showed higher C/N ratio probably because of the dissolution of carbon-rich plant materials.     

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.     

Purification and characterization of cathepsin L from the white muscle of chum salmon, Oncorhynchus keta

1. Cathepsin L of the white muscle of chum salmon (Oncorhynchus keta) in spawning migration was purified to homogeneity by a series of chromatography on DEAE-Sephadex (1st), SP-Sephadex, CM-Sephadex, DEAE-Sephadex (2nd) and Sephadex G-100. 2. The molecular weight of salmon muscle cathepsin L was estimated to be 30,000 and its isoelectric point was 5.2. 3. Cathepsin L had a pH optimum of 5.6, required a thiol-reducing reagent for activation, and was inhibited by cysteine protease inhibitors. 4. The Km and kcat values for Z-Phe-Arg-MCA were determined to be 1.68 microM and 15.8 s-1, respectively. This enzyme hydrolyzed proteins such as insulin B chain, hemoglobin, serum albumin and azocasein easily. 5. The bond specificity to oxidized insulin B chain inferred that the enzyme had a preference for hydrophobic amino acid in P2 and P3 residues.     

Occurrence of VIP and peptide HM in human pancreas and their influence on pancreatic endocrine secretion in man

By immunohistochemistry it was found that VIP- and peptide HI/peptide HM (PHI/PHM)-like immunoreactivity occurred in autonomic neurons in the human pancreas. Antisera against both VIP and PHI/PHM reacted with neuronal cells in local ganglia and these ganglia also contained PHI/PHM- and VIP-immunoreactive fibre plexuses. VIP- and PHI/PHM-positive fibres were also seen close to the Langerhans' islets. In addition, PHI/PHM- but not VIP-like immunoreactivity was observed in the endocrine cells often located in the periphery of the islets. The nature of these PHI/PHM-positive cells remains to be established. I.v. infusion of VIP at constant rates of 300 and 900 pmol/kg X h for 30 min in 6 healthy volunteers resulted in plateau values of 102 +/- 26 and 291 +/- 25 pM, respectively. These levels of VIP which are above those found in the circulation under physiological conditions stimulated secretion of insulin, C-peptide and pancreatic glucagon dose-dependently. On the contrary prolonged (60 min) infusion of PHM in doses resulting in plasma levels up to 1340 +/- 405 pM had no effect on pancreatic hormone secretion. These findings suggest that VIP is a likely neurotransmitter in the control of endocrine pancreatic secretion while PHM has a less prominent role, if any.