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.     

Quantitative detection of bisphenol A and bisphenol A diglycidyl ether metabolites in human plasma by liquid chromatography–electrospray mass spectrometry

Due to the ubiquity of epoxy resin compounds and their potential role in increasing the risk for reproductive dysfunction and cancer, the need for an assessment of human exposure is urgent. Therefore, we developed a method for measuring bisphenol A (BPA) and bisphenol A diglycidyl ether (BADGE) metabolites in human blood samples using high-performance liquid chromatography–electrospray ionization mass spectrometry (LC–MS). Human blood samples were processed using enzymatic deconjugation of the glucuronides followed by a novel sample preparation procedure using a solid-phase-cartridge column. This selective analytical method permits rapid detection of the metabolites, free BPA and a hydrolysis product of BADGE (BADGE-4OH) with detection limits in the low nanogram per milliliter range (0.1 ng ml⁻¹ of BPA and 0.5 ng ml⁻¹ of BADGE-4OH). The sample extraction was achieved by Oasis HLB column on gradient elution. The recoveries of BPA and BADGE-4OH added to human plasma samples were above 70.0% with a standard deviation of less than 5.0%. This selective, sensitive and accurate method will assist in elucidating potential associations between human exposure to epoxy-based compounds and adverse health effects.     

Influence of rainfall on sleeping site choice by a group of anubis baboons (Papio anubis)

For diurnal nonhuman primates, shifting among different sleeping sites may provide multiple benefits such as better protection from predators, reduced risk of parasitic infection, and closer proximity to spatially and temporally heterogeneous food and water. This last benefit may be particularly important in sleeping site selection by primates living in savanna‐woodlands where rainfall is more limited and more seasonally pronounced than in rainforests. Here, we examined the influence of rainfall, a factor that affects food and water availability, on the use of sleeping sites by anubis baboons (Papio anubis) over two 13‐month study periods that differed in rainfall patterns. We predicted that during wet periods, when food and water availability should be higher, the study group would limit the number of sleeping sites and would stay at each one for more consecutive nights than during dry periods. Conversely, we predicted that during dry periods the group would increase the number of sleeping sites and stay at each one for fewer consecutive nights as they searched more widely for food and water. We also predicted that the group would more often choose sleeping sites closer to the center of the area used during daytime (between 07:00 and 19:00) during wet months than during dry months. Using Global Positioning System data from collared individuals, we found that our first prediction was not supported on either monthly or yearly timescales, although past monthly rainfall predicted the use of the main sleeping site in the second study period. Our second prediction was supported only on a yearly timescale. This study suggests that baboons’ choice of sleeping sites is fluid over time while being sensitive to local environmental conditions, one of which may be rainfall.     

Effects of intervals between jumps or bouts on osteogenic response to loading.

Prolonged loading repetitions can diminish the mechanosensitivity of bones, and increased intervals between loading might restore sensitivity. This study was designed to investigate the effects of intervals between loadings or bouts on osteogenic response. Forty female Fisher 344 rats aged 5 wk were divided into a control group and three exercise groups: 20 jumps in a single bout with a 3-s (S3) or 30-s (S30) jump interval, or 20 jumps in 2 bouts (10 x 2) separated by a 6-h interval with a 3-s jump interval (D3). After 8 wk of training, the bone masses per body weight of the femur and tibia were significantly greater in the three exercise groups than in the control group, and these values were also greater in S30 than in S3, although they were at the same level in D3 and S3. These data suggest that a longer interval (30 s) between individual loading had more effective anabolic effects on bone than a shorter interval (3 s).     

Effects of Exogenous Amino Acids on Iron Uptake in Relation to their Effects on Photoperiodic Flowering in Lemna paucicostata 6746

The flowering of Lemna paucicostata 6746 grown on 14-h photoperiodwas enhanced by the addition of high concentrations of ironto the medium, which also increased the endogenous iron concentration.The addition of asparagine, aspartate, glutamate, -alanine,glycine or serine to the medium also increased the endogenousiron level, resulting in the promotion of flowering. In contrast,the addition of cysteine, cystine, glutamine, arginine, threonineor phenylalanine lowered the endogenous iron level, resultingin the inhibition of flowering. Glycine and asparagine added to the medium during an inductive96-h dark period did not promote iron uptake and had no effecton flowering, but when added during the subsequent 120-h lightperiod, they promoted both iron uptake and flowering response.The increase in the endogenous iron level seems to favor floraldevelopment rather than induction of photoperiodic floweringof Lemna paucicostata 6746. (Received September 8, 1986; Accepted March 31, 1987)     

Establishment of a human T cell hybridoma cell line producing suppressor factor specific for anti-thyroglobulin antibody production

Thyroglobulin (Tg)-binding peripheral blood T cells from a normal individual were fused with a T cell leukemia cell line (Jurkat-AG9) treated by emetine and actinomycin D. Several cell lines were established from thus-prepared human T cell hybridomas. The culture supernatant from one of these lines (Tg-Ts47) whose phenotype was OKT3- 11+ 4+ 8- suppressed the generation of Tg-specific antibody-forming cells from the lymphocytes of patients with Hashimotos' chronic thyroiditis, but not anti-SRBC and anti-ovalbumin antibody production from both autologous and patient lymphocytes. Tg-Ts47-derived factors also bore Tg antigen-binding sites. The suppressive activity of the supernatants was shown in almost all patients lymphocytes tested. This indicated that the supernatants of Tg-Ts47 line contain a suppressive factor specific for Tg antigen and capable of acting across allogeneic barriers.     

A case of von Recklinghausen's disease associated with pheochromocytoma and papillary carcinoma of the thyroid gland

A 58-year-old woman was admitted to our hospital complaining of headache, dizziness and intermittent elevation of blood pressure. Multiple café-au-lait spots and neurofibromas had appeared on the back and the limbs since the age of 30 years. At the age of 54 years she underwent total thyroidectomy because of papillary carcinoma of the thyroid gland. On admission, the levels of plasma norepinephrine and epinephrine, urinary norepinephrine and normetanephrine were all within the normal range. However, urinary excretion of metanephrine was markedly increased to 1.49 +/- 0.45 (Mean +/- SD) mg/day and that of epinephrine was also slightly increased. The computed tomographic scans of the abdomen and the scintigraphy with 131I-metaiodobenzylguanidine revealed a tumor mass in the region of the right adrenal gland. The tumor was histologically confirmed to be pheochromocytoma at the operation. In her family history, her mother and one of her two sisters had von Recklinghausen's disease and another sister suffered from follicular carcinoma of the thyroid gland. As far as we know, this paper is the first report of a patient with von Recklinghausen's disease associated with both pheochromocytoma and non-medullary carcinoma of the thyroid gland, and her family.