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.     

Selective diapedesis of Th1 cells induced by endothelial cell RANTES.

Differentiated CD4 T cells can be divided into Th1 and Th2 types based on the cytokines they produce. Differential expression of chemokine receptors on either the Th1-type or the Th2-type cell suggests that Th1-type and Th2-type cells differ not only in cytokine production but also in their migratory capacity. Stimulation of endothelial cells with IFN-gamma selectively enhanced transmigration of Th1-type cells, but not Th2-type cells, in a transendothelial migration assay. Enhanced transmigration of Th1-type cells was dependent on the chemokine RANTES produced by endothelial cells, as indicated by the findings that Ab neutralizing RANTES, or Ab to its receptor CCR5, inhibited transmigration. Neutralizing Ab to chemokines macrophage-inflammatory protein-1alpha or monocyte chemotactic protein-1 did not inhibit Th1 selective migration. Whereas anti-CD18 and anti-CD54 blocked basal levels of Th1-type cell adherence to endothelial cells and also inhibited transmigration, anti-RANTES blocked only transmigration, indicating that RANTES appeared to induce transmigration of adherent T cells. RANTES seemed to promote diapedesis of adherent Th1-type cells by augmenting pseudopod formation in conjunction with actin rearrangement by a pathway that was sensitive to the phosphoinositol 3-kinase inhibitor wortmannin and to the Rho GTP-binding protein inhibitor, epidermal cell differentiation inhibitor. Thus, enhancement of Th1-type selective migration appeared to be responsible for the diapedesis induced by interaction between CCR5 on Th1-type cells and RANTES produced by endothelial cells. Further evidence that CCR5 and RANTES play a modulatory role in Th1-type selective migration derives from the abrogation of this migration by anti-RANTES and anti-CCR5 Abs.     

Localization of carbonic anhydrase in the rat lung

Summary The localization of carbonic anhydrase in the rat lung has been demonstrated, at light and electron microscopic levels, by the cobalt bicarbonate histochemical method of Hansson. Focal deposits of the cobalt sulfide reaction product were found not only in the capillary endothelium of the alveolar walls, but also in the small and large alveolar cells. The histochemical reaction was abolished by two potent inhibitors, acetazolamide (10^–5 to 10^–6 M) and KCNO (5×10^–3 to 10×10^–3 M). Physiological assay with Maren's method indicated that values for carbonic anhydrase activity in rat lung are 4.4±0.8 UA/mg of protein, 25.0±5.5 UA/mg of nitrogen, and 369±86 UA/g of wet weight. In addition, it was calculated that after fixation in glutaraldehyde-formaldehyde-picric acid about 9% activity is retained.     

The calcium-binding property of equine lysozyme

It was found that equine lysozyme binds one Ca2+. It was eluted with equimolar Ca2+ from a Bio-Gel P-4 column. Human lysozyme did not behave similarly. Equine lysozyme is concluded to be a calcium metallo-protein like alpha-lactalbumin, which is a homologue of hen egg white lysozyme.     

Division and gametophytic tissue formation from protoplasts of young sporophytes in fern Lygodium japonicum

Protoplasts isolated enzymatically from juvenile leaflets of sporophytes in fern Lyqodium japonicum were inoculated in a MS medium containing NAA 2.7 m, BAP 2.2 m, 0.6 M mannitol, and 0.05 M sucrose. Cell division took place within 8 days of culture initiation, and after 30 days cell-clusters with 10–15 cells were observed. When the cell-clusters were transferred into fresh hormone-free medium containing no mannitol, they developed rhizoids and protonema-like regenerants. About 2 months later, cordate prothallia developed bearing both antheridia and archegonia.Abbreviations MS Murashige and Skoog (1962) - NAA naphthalene acetic acid - BAP 6-benzylaminopurine     

Behavior of chloroplasts and chloroplast nuclei during spermatogenesis in the fern,Pteris vittata L.

Summary The fate of the chloroplasts and chloroplast nuclei (cp-nuclei) was followed during spermatogenesis in the fernPteris vittata L. by epifluorescence microscopy after staining with 4-6-diamidino-2-phenylindole (DAPI) and by quantitation of chloroplast DNA (cp-DNA) by fluorimetry using a video intensified microscope photon counting system (VIMPICS). The spores were grown on solid medium that contained antheridiogen (An_ptd), and formed an antheridium initial on the protonema cell. The antheridium initial divided and produced 16 spermatocytes and 3 surrounding cells. The chloroplasts in the spermatocytes decreased in volume as cell division was repeated, until finally the volume of each chloroplast was 1/15 of that of the primary chloroplasts. The DNA content of the chloroplasts was also reduced to 1/5 of the original value and when the sperm matured, the fluorescence of cp-DNA disappeared. In the 16-cell spermatocyte, the recognition of the fluorescence of chlorophyll in the chloroplasts with a green excitation filter became difficult. But, the plastids could be observed until the final stage of the sperm. From these observations, it appears that there are two steps in the metamorphosis of chloroplasts during spermatogenesis in the fern. The first step involves the decrease in the volume of chloroplasts, accompanied by reduction of the DNA content, and the second step involves the change of the physical state of chloroplasts to amyloplasts and the disappearance of the cp-DNA from the amyloplasts.     

Contribution of the 6-120 disulfide bond of alpha-lactalbumin to the stabilities of its native and molten globule states.

The unfolding and refolding of a derivative of alpha-lactalbumin, in which the disulfide bond between Cys6 and Cys120 is selectively reduced and S-carboxymethylated, are investigated by equilibrium and kinetic circular dichroism measurements. The native conformation of this derivative is known to be essentially identical to that of intact alpha-lactalbumin. The equilibrium unfolding of the derivative involves a stable intermediate, which is also similar to the molten globule state of the disulfide intact protein. The results of stopped-flow circular dichroism experiments show that the same intermediate is formed rapidly as a transient intermediate in kinetic refolding. The conformational stabilities for the native and intermediate states have been estimated and compared with the stabilities for the corresponding states of intact alpha-lactalbumin. The stabilization of the native state by the disulfide has been interpreted in terms of a decrease in chain entropy in the unfolded state and elimination of the strain imposed on the disulfide bond in the native state. The molten globule state is also stabilized by the disulfide bond, although the degree of stabilization of the molten globule state is smaller than of the native state. The results suggest that, in the molten globule state, some ordered structures are present within the loop moiety formed by the 6-120 disulfide.     

Mutations in the M1 region of the nicotinic acetylcholine receptor alter the sensitivity to inhibition by quinacrine

Summary 1. Site directed mutagenesis was used to alter the structure ofTorpedo californica nicotinic acetylcholine receptor (nAChR) and to identify amino acid residues which contribute to noncompetitive inhibition by quinacrine. Mutant receptors were expressed inXenopus laevis oocytes injected within vitro synthesized mRNA and the whole cell currents induced by acetylcholine (ACh) were recorded by two electrode voltage clamp.2. A series of mutations of a highly conserved Arg at position 209 of the subunit ofTorpedo californica nAChR revealed that positively charged amino acids are required for functional receptor expression. Mutation of Arg to Lys (R209K) or His (R209H) at position 209 shifted the EC₅₀ for ACh slightly from 5µM to 12µM and increased the normalized maximal channel activity 8.5-and 3.2-fold, respectively.3. These mutations altered the sensitivity of nAChR to noncompetitive inhibition by quinacrine. The extent of inhibition of ion channel function by quinacrine was decreased as pH increased in both wild type and mutant nAChR suggesting that the doubly charged form of quinacrine was responsible for the inhibition.4. Further mutations at different positions of the subunit suggest the contribution of Pro and Tyr residues at positions 211 and 213 to quinacrine inhibition whereas mutationsI210A andL212A did not have any effects. None of these mutations changed the sensitivity of nAChR to inhibition by a different noncompetitive inhibitor, chlorpromazine.5. These findings support a hypothesis that the quinacrine binding site is located in the lumen of the ion channel. In addition, the quantitative effect of point mutations at alternate positions on the sensitivity of quinacrine inhibition suggests that the secondary structure at the beginning of M1 region might be sheet structure.