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.     

Cleavage of double helical DNA by Cu2+ ion in the presence of bisintercalator containing penta(ethylene glycol) connector chain

In designing new DNA recognizing and cleaving reagents, we introduce herein a bisacridine derivative (referred to as bisacridine) in which two acridine heterocycles are connected by a penta(ethylene glycol) bridging chain. This compound offers two possible functions: 1, stabilization of DNA bisacridine intercalator complex by metal ion. The penta(ethylene glycol) chain stabilizes metal ions binding to the phosphate site of DNA, where the penta(ethylene glycol) chain constitutes a part of a pseudomacrocyclic ligand for metal binding; and 2, enhancement of metal-assisted hydrolytic cleavage of DNA by means of a metal concentration effect by the pseudomacrocyclic ethereal chain. The binding isotherms of bisacridine with DNA in the presence of metal ions showed that the binding was mainly governed by the cation exchange reaction on the anionic DNA polymer chain, i.e., the exchange between metal ions and the cationic bisacridine. The bisacridine showed an increase DNA binding ability compared to quinacrine, the monoacridine counterpart, and caused an enhancement of DNA cleavage in the presence of Cu2+ ions. Additional experiments which included DNase 1 footprinting in the presence of bisacridine and the DNA cleavage by Cu2+/bisacridine using a 32P end-labelled DNA fragment, suggested that the Cu2(+)-assisted DNA cleavage sites in the presence of bisacridine were in reasonable overlap with the DNA binding sites of bisacridine.     

A newly established human acute lymphoblastic leukemia cell line with characteristics of the earliest B-cell maturation

A new human acute lymphoblastic leukemia (ALL) cell line, designated HBL-3, was established from the bone marrow of a patient with non-T-ALL. The HBL-3 cell line expressed B4 (CD 19), BA-1 (CD 24) and HLA-DR antigens, but not surface immunoglobulin (SIg) or cytoplasmic immunoglobulin (CIg). The cell line lacked the common acute lymphoblastic leukemia antigen (CALLA) and antigenic markers characteristic of T-cell and myeloid cell lineages. The HBL-3 cells had structural rearrangements of both the homologous chromosome 9s, including a translocation with chromosome 1 which has been reported in a patient with common ALL. The cell line had rearranged immunoglobulin heavy chain genes but retained germ-line κ light chain genes and germ-line T-cell receptorβ- and γ-chain genes. The HBL-3 cell line was strongly positive for terminal deoxynucleotidyl transferase (TdT). These findings indicate that the HBL-3 cell line is derived from the earliest B-cell committed to B-cell lineage.     

Establishment of a monoclonal antibody recognizing cyclobutane-type thymine dimers in DNA: a comparative study with 64M-1 antibody specific for (6-4)photoproducts

We obtained a monoclonal antibody (TDM-1) binding to 313-nm UV-irradiated DNA in the presence of acetophenone. The binding of TDM-1 to 254-nm UV-irradiated DNA was not reduced with the subsequent irradiation of 313-nm UV. Furthermore, the treatment of UV-irradiated DNA with photolyase from E. coli and visible light exposure reduced both the antibody binding and the amount of thymine dimers in the DNA. A competitive inhibition assay revealed that the binding of TDM-1 to UV-irradiated DNA was inhibited with photolyase, but not with 64M-1 antibody specific for (6-4)photoproducts. These results suggest that TDM-1 antibody recognizes cyclobutane-type thymine dimers in DNA. Using TDM-1 and 64M-1 antibodies, we differentially measured each type of damage in DNA extracted from UV-irradiated mammalian cells. Repair experiments confirm that thymine dimers are excised from UV-irradiated cellular DNA more slowly than (6-4)photoproducts, and that the excision rates of thymine dimers and (6-4)photoproducts are lower in mouse NIH3T3 cells than in human cells.     

Cell-specific properties of red cell and liver ferritin from bullfrog tadpoles probed by phosphorylation in vitro

Cell-specific differences occur in the primary structure of ferritin. For example, red cell and liver ferritin from bullfrog tadpoles differ by 1.5 times in serine content. To determine if cell-specific differences in ferritin primary structure are expressed in the tetraeicosomer, which thus might distinguish the proteins in a functional state, phosphorylation in vitro was employed as a probe using [gamma-32P]ATP and the catalytic subunit from the cAMP-dependent protein kinase of bovine skeletal muscle. Subunits of both proteins in the tetraeicosomers were phosphorylated. Based on tryptic peptide maps, five regions common to both red cell and liver apoferritin were phosphorylated, as confirmed for two peptides by amino acid analyses. [32P]Apoferritin from red cells yielded an additional four 32P-fragments by mapping, at least three of which were unique by amino acid analysis and, in one case, might represent a 32P-Fe complex bound by a fragment of the iron-binding site. One peptide appeared to be unique to liver apoferritin. High concentrations of ATP yielded one additional peptide common to liver and red cell and one red cell-specific peptide in the tryptic peptide maps. The maximum moles of 32P/molecule were 13 +/- 4 and 6 +/- 2, respectively, for red cell and liver apoferritin, which corresponded within experimental error to the number of 32P-tryptic peptides. The level of phosphorylation was, on the average, not more than one site/subunit. Furthermore, above certain levels of phosphorylation, some subunits in the assemblage of 24 appeared to be unavailable as substrates, possibly because of charge repulsion or conformational changes. The possibility that post-translational modifications of ferritin which amplify cell-specific structural features occur in vivo with cytoplasmic components, e.g. protein kinases, is considered in terms of the physiological availability of iron from different iron storage cells and developmental changes in iron storage.     

Lipase modulator protein (LimL) of Pseudomonas sp. strain 109.

Plasmids containing a Pseudomonas sp. strain 109 extracellular lipase gene (lipL) lacking NH2-terminal sequence and a lipase modulator gene (limL) lacking the NH2-terminal hydrophobic region were constructed and expressed independently in Escherichia coli by using the T7 promoter expression vector system. Recombinant LipL (rLipL) was produced as inclusion bodies, whereas recombinant LimL (rLimL) was present as a soluble protein. During in vitro renaturation of the purified rLipL inclusion bodies after they had been dissolved in 8 M urea, addition of rLimL was essential to solubilize and modulate rLipL. The solubility and activity of rLipL were influenced by the rLimL/rLipL molar ratio; the highest level of solubility was obtained at an rLimL/rLipL ratio of 4:5, whereas the highest activity level was obtained at an rLimL/rLipL ratio of 4:1. After renaturation, rLipL and rLimL were coprecipitated with anti-rLipL antibody, indicating the formation of an rLipL-rLimL complex. Activity of the native lipase purified from Pseudomonas sp. strain 109 was also inhibited by rLimL. By Western blotting (immunoblotting) with anti-rLimL antibody, native LimL was detected in Pseudomonas cells solubilized by sarcosyl treatment. LimL was purified from Pseudomonas sp. strain 109, and the NH2-terminal amino acid sequence was determined to be NH2-Leu-Glu-Pro-Ser-Pro-Ala-Pro-. We propose that to prevent membrane degradation, LimL weakens lipase activity inside the cell, especially in the periplasm, in addition to modulating lipase folding.     

Mouse {beta}-galactoside {alpha}2,3-sialyltransferases: comparison of in vitro substrate specificities and tissue specific expression

Four types of ß-galactoside