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.     

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.     

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.     

Localization of neutrophil adherence-inhibiting factor in guinea pig platelets

The effect of constituents of guinea pig platelets on neutrophil adherence was examined. The platelet sonicate supernatant contained adherence-inhibiting activity which strongly inhibited neutrophil adherence to glass. When the platelet sonicate supernatant was treated with neuraminidase or trypsin, the adherence-inhibiting activity was significantly inhibited, suggesting that the adherence-inhibiting factor (AIF) is a glycoprotein. The subcellular fractionation experiments indicated that the AIF activity was present at about 40% in both the cytosol and granule fractions. From the Sephadex G-200 gel filtration analysis, AIF of cytosol fraction and granule fraction proved to be different molecules, with molecular masses of about 230 and 12 kDa, respectively. When platelets were stimulated with thrombin, about 20% of total AIF was released extracellularly without the release of the cytoplasmic enzyme lactate dehydrogenase. These results suggest the possibility that a biologically active substance, AIF, is released from platelets in response to stimuli and regulates neutrophil functions through interference with neutrophil adherence.     

Immune response to Toxoplasma gondii--analysis of suppressor T cells in a patient with symptomatic acute toxoplasmosis

Unresponsiveness of antigen-dependent (Toxoplasma-specific and purified protein derivative of tuberculin [PPD]-specific) T-cell proliferative responses of peripheral blood leukocytes (PBL) was observed in a patient with symptomatic acute toxoplasmosis. The immunosuppression of T-cell responses was mediated by Leu 1+, Leu 2a+, and Leu 3a- suppressor T cells that were induced by Toxoplasma gondii antigen and suppressed both Toxoplasma-specific and PPD-specific PBL T-cell responses from a patient with chronic toxoplasmosis when PBL of these patients were mixed and cocultured in vitro. Participation of class II molecules of HLA in Toxoplasma-specific proliferative T-cell responses and activation of suppressor T cells was examined by using monoclonal antibodies specific for HLA-DR and HLA-DQ molecules. Anti-HLA-DQ monoclonal antibody released the suppressive activity, while anti-HLA-DR monoclonal antibody inhibited Toxoplasma-specific T-cell responses. Thus, the suppressive effect of PBL from a patient with acute toxoplasmosis on antigen-dependent PBL T-cell responses from a patient with chronic toxoplasmosis was mediated by HLA-DQ molecules. By contrast, Toxoplasma-specific T-cell responses were activated by HLA-DR molecules (presumably present on antigen-presenting cells).