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.     

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.     

Nitrogen Uptake and Plant Growth II. An Empirical Model of Vegetative Growth and Partitioning

An empirical model of vegetative plant growth is presented.The model is based on experimental data on Polygonum cuspidatum,which showed (1) that the partitioning of dry matter and nitrogenamong organs was linearly related to the nitrogen concentrationof the whole plant and (2) that leaf thickness was negativelycorrelated with leaf nitrogen concentration. The model properlydescribes the behaviour of plants. Steady-state solutions ofthe model give the relative growth rate, specific leaf weight,and partitioning of dry matter and nitrogen among organs withthe net assimilation rate and the specific absorption rate asenvironmental variables. The effect of nitrogen removal on drymatter and nitrogen partitioning was examined as non-steady-statedynamic solutions of the model. The model predicted not onlyreduced leaf growth and enhanced root growth but also a fluxof nitrogen from the leaf to the root, which agreed with theexperimental results. Mathematical model, partitioning of dry matter and nitrogen, plant nitrogen, relative growth rate, shoot: root ratio, specific leaf weight     

Synchronization of Mating Reactivity Rhythms in Populations of Paramecium bursaria

Cell populations of Paramecium bursaria show arhythmic mating reactivity after exposure to constant light (LL) for more than 2 wk. After this arhythmic population is exposed to darkness for 9 h, the mating reactivity rhythm of the cell population reappears. The phases of rhythms in individual cells are synchronized to each other. When the arhythmic population in constant light is exposed to dark pulses of various durations, the first peak of the recovered mating reactivity rhythm appears 6 h after the end of the dark pulse. Thus, in the case of dark pulses to cells in LL, the transition from dark to light sets the phase of the subsequent mating reactivity rhythm. When an arhythmic population in LL is transferred to constant darkness (DD), a rhythm of mating reactivity also appears and, in this case, the first peak of the rhythm occurs 18 h after the LL to DD transition. Therefore, arhythmic populations of cells in LL can be synchronized by either a dark pulse or by transition to continuous darkness. When the arhythmic populations in LL were transferred to various light/dark (LD) cycles, the mating reactivity rhythms entrained to LD cycles of 18 to 30 h in duration. Finally, mating rhythms can also be synchronized by treatment with puromycin (400 μg/ml for 6–18 h).     

Retrotransposon-induced ectopic expression of the Om(2D) gene causes the eye-specific Om(M) phenotype in Drosophila ananassae

Optic morphology (Om) mutations in Drosophila ananassae map to at least 22 loci, which are scattered throughout the genome. Om mutations are all semidominant, neomorphic, nonpleiotropic, and associated with the insertion of a retrotransposon, tom. We have found that the Om(2D) gene encodes a novel protein containing histidine/proline repeats, and is ubiquitously expressed during embryogenesis. The Om(2D) RNA is not detected in wild-type eye imaginal discs, but is abundantly found in the center of the eye discs of Om(2D) mutants, where excessive cell death occurs. D. melanogaster flies transformed with the Om(2D) cDNA under control of the hsp70 promoter display abnormal eye morphology when heat-shocked at the third larval instar stage. These results suggest that the Om(2D) gene is not normally expressed in the eye imaginal discs, but its ectopic expression, induced by the tom element, in the eye disc of third instar larvae results in defects in adult eye morphology.     

Selective Adhesion of Thiobacillus ferrooxidans to Pyrite

Bacterial adhesion to mineral surfaces plays an important role not only in bacterial survival in natural ecosystems, but also in mining industry applications. Selective adhesion was investigated with Thiobacillus ferrooxidans by using four minerals, pyrite, quartz, chalcopyrite, and galena. Escherichia coli was used as a control bacterium. Contact angles were used as indicators of hydrophobicity, which was an important factor in the interaction between minerals and bacteria. The contact angle of E. coli in a 0.5% sodium chloride solution was 31°, and the contact angle of T. ferrooxidans in a pH 2.0 sulfuric acid solution was 23°. E. coli tended to adhere to more hydrophobic minerals by hydrophobic interaction, while T. ferrooxidans selectively adhered to iron-containing minerals, such as pyrite and chalcopyrite. Ferrous ion inhibited the selective adhesion of T. ferrooxidans to pyrite competitively, while ferric ion scarcely inhibited such adhesion. When selective adhesion was quenched by ferrous ion completely, adhesion of T. ferrooxidans was controlled by hydrophilic interactions. Adhesion of E. coli to pyrite exhibited a liner relationship on langmuir isotherm plots, but adhesion of T. ferrooxidans did not. T. ferrooxidans recognized the reduced iron in minerals and selectively adhered to pyrite and chalcopyrite by a strong interaction other than the physical interaction.     

Dual Appearances of Pigment Cells from In Vitro Cultured Embryonic Cells of Japanese Flounder: An Implication for a Differentiation–Associated Clock

The cells dissociated from developing embryos of Japanese flounder (Paralichthys olivaceus) are cultured in vitro to examine the developmental fate of their pigment cells in relation to establishment of bilaterally asymmetric integumental coloration in vivo. When neurula embryos are dissociated using trypsin–EDTA in Dulbecco's modified Ca²⁺–, Mg²⁺–free phosphate buffered saline and then cultured in vitro using L–15–based fetal calf serum–supplemented growth medium at 20°C, numerous pigment cells appear twice in the same culture with an interval of approximately 1 month even under similar culture conditions. The first group of pigment cells, which is relatively larger in cell size (about 70 μm wide) and lower in cell density, emerges within 12 hr after plating, whereas the second, which is far smaller in cell size (about 30 μm) and overwhelmingly higher in cell density than the first, does so about 1 month after plating. The timing of their appearances in vitro is in good accordance, respectively, with that observed for the larvae under normal development in vivo; the first group appears at the period corresponding to hatching, whereas the second at the period corresponding to the completion of metamorphosis. Light microscopic examinations disclose that each group of pigment cells is composed of black melanophores and reflecting leucophores, and that the population density of melanophores and leucophores in the first group at the climax of appearance is approximated as 1:4. Typical xanthophores that are distributed in the skin of the larvae of this species are scarcely observed in culture in vitro. Because of their dual synchronous appearances with about 1 month interval under the similar culture conditions, and because of their low proliferative activity during the periods from the first appearance to the second, it is presumed that both groups of pigment cells are installed with a clock set differently for their differentiation. Light and electron microscopic immunocytochemistry on cultured cells using the HNK–I antibody, which marks avian migratory neural crest cells, both disclose that the antibody cross–reacts with all these pigment cells, and that a certain number of immunoreactive unpigmented cells exist even at the time of the second appearance of pigment cells. These findings would imply that the second group of pigment cells served in a form of undifferentiated propigment cells up to metamorphosis, at which they start to differentiate under control of a clock presumably set during neurulation.