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.     

Production,Isolation and Pharmacological Studies of AI-77s

Two potent gastroprotective substances against experimental gastric ulcers in rats induced by stress and five of their analogues were isolated from the culture broth of bacterial strain AI-77 which was classified taxonomically as Bacillus pumilus. Physico-chemical properties and pharmacological activities of the seven compounds were examined and compared with each other.     

Electrophoretic Studies on Plant Protoplasts I. pH Dependence of Zeta Potentials of Protoplasts from Various Sources

Electrophoretic mobilities of plant protoplasts from varioussources were measured, as a function of the pH of the medium,by a micro-electrophoresis technique to characterize the protoplastsin terms of curves of zeta potential vs. protoplast surfacepH (pH_s). The shape of the curves of zeta potential vs. pH_scurves differed among preparations of protoplasts isolated fromvarious species and strains. The isoelectric points (pI) ofthe protoplasts measured in this study were between 3.0 and4.0. These differences among the protoplasts suggest that itmay be possible to develop an electrophoretic method for theselection of protoplasts. The shape of the curves of zeta potentialvs. pH_s also indicated that carboxyl groups, as well as phosphategroups, may contribute to the negative charges on the surfaceof protoplasts. (Received October 14, 1988; Accepted February 22, 1989)     

Endothelin-3 stimulates the hypothalamic-pituitary-adrenal axis.

Endothelin-3 (ET-3) is a member of the novel vasoconstrictive peptide family, identified in porcine central nervous system. Intravenous bolus injection of 1000 pmol/kg of ET-3 in freely moving rats caused significant increases in plasma ACTH and corticosterone levels, almost equivalent to those of 100 pmol/kg of rat corticotropin-releasing hormone (rCRH). The action of ET-3 was virtually abolished by pretreatment of CRH-antagonist, alpha-helical CRH. When ET-3 was added to cultured anterior pituitary cells, neither direct stimulation of ACTH release nor potentiation of rCRH action was noted. The results indicate that ET-3 may function as a neuropeptide and stimulation of the CRH-neurons, direct or inderect, is mainly responsible for activation of ACTH and corticosterone release.     

Characterization of antisera to 2-hydroxyestradiol and 4-hydroxyestradiol using 6-(O-carboxymethyl)oxime- and 17-hemisuccinate-bovine serum albumin conjugates and radioimmunoassay

For radioimmunoassay of the catechol estrogens, four hapten-bovine serum albumin (BSA) conjugates were prepared from 6-oxo-2-hydroxyestradiol 6-(O-carboxymethyl)oxime, 2-hydroxyestradiol 17-hemisuccinate, 6-oxo-4-hydroxyestradiol 6-(O-carboxymethyl)oxime and 4-hydroxyestradiol 17-hemisuccinate by coupling with BSA, employing the mixed anhydride method. The antisera elicited in rabbits by immunization with these antigens showed high affinity and specificity for 2-hydroxyestradiol or 4-hydroxyestradiol with cross-reactivities to a few structurally related estrogens. The specificity of antisera obtained is discussed in relation to the site of attachment of the hapten to BSA.     

On the relationship between two indices (“Bulk density” and “dry-matter content”) of dry-matter accumulation in plant organs

“Bulk density” and “dry-matter content” are useful indices of dry-matter accumulation in plant organs. A theoretical equation describing the relationship between these two indices was put forward. To examine the reliability of this equation, the seasonal changes of these two indices were investigated in the leaves and stems of different ages ofAucuba japonica. In each organ both indices varied seasonally almost parallel to each other, but the seasonal changes of dry-matter content were less obvious than those of bulk density. The observed bulk density was always larger than that calculated from the observed dry-matter content by the theoretical equation. A drying experiment showed that this discrepancy was caused by the decrement of the volume of the plant material by water loss during the period from the weight measurement to the volume measurement. When the water loss was negligible, the equation described well the relationships between the two indices of the experimental materials. It was also shown that this equation was useful for the estimation of the amount of air space in plant materials.     

Sensitivity of mice to systemic hyperthermia: effect of the transplantation of ascites tumor cells modified by preheating, and by an injection of CDDP or interferon

Effects of preheating and injection of cis-DDP (CDDP) or interferon on tumor-induced sensitization to systemic hyperthermia (SH) was investigated in mice. LD50 of SH at 42.0 +/- 0.2 degrees C (core body temperature) was 43 min in normal mice and 8 min in mice which were i.p. transplanted with FMA3 cells at a dose of 10(5) one day before. In mice which had received the SH for 10 min one hour before, one hour after or one day before the transplantation, LD50 of the SH one day after the transplantation was 41, 35 and 22 min, respectively. An injection of CDDP given i.p. at a dose of 4 mg/kg one day after the transplantation, which was effective to kill about 99% of the tumor cells, did not change the course of thermosensitization after the transplantation. An i.p. injection of mouse interferon did not change the thermosensitivity of normal mice, but greatly suppressed the thermosensitizing effect of tumor cells when it was given one day before the transplantation.