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.     

The Gene Encoding Flavanone 3-Hydroxylase is Expressed Normally in the Pale Yellow Flowers of the Japanese Morning Glory Carrying the speckled Mutation Which Produce Neither Flavonol nor Anthocyanin but Accumulate Chalcone, Aurone and Flavanone

The Japanese morning glory carrying the recessive mutable speckledallele with the dominant speckled-activator bears colorlessflowers with fine and round colored spots distributed over thecorolla whereas the plant without the speckled-activator producespale yellow flowers. Previous chemical analysis has indicatedthat a mutation in the gene for flavanone 3-hydroxylase (F3H)is a likely candidate for the speckled allele. However, theF3HmRNA without sequence alteration accumulates normally inthe pale yellow flowers, indicating that the speckled alleleis neither the F3H gene nor a regulatory gene acting on theF3H gene expression. (Received April 4, 1997; Accepted June 2, 1997)     

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.     

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).     

A new gene controlling the frequency of cell division per round of DNA replication in Escherichia coli

Summary A novel mutant of Escherichia coli, named cfcA1, was isolated from a temperature-sensitive dnaB42 strain, and found to have the following characteristics. Division arrest and lethality induced by inhibition of DNA replication was reduced and delayed in the cfcA1 dnaB42 strain, as compared with the parental dnaB42 strain. Two types of inhibition of division induced by the addition of nalidixic acid or hydroxyurea were suppressed by the cfcA1 mutation. Under permissive conditions for DNA replication, the colony forming ability of cfcA1 cells was significantly reduced as compared with that of cfc ⁺ cells; conversely the division rate of cfcA1 cells was higher than that of cfc ⁺ cells. The cfcA1 mutation partially restored division arrest induced in the thermosensitive ftsZ84 mutant at the restrictive temperature and suppresed the UV sensitivity of the lon mutation. The mutation was mapped at 79.2 min on the E. coli chromosome. Taking these properties into account, it is hypothesized that the cfcA gene is involved in determining the frequency of cell division per round of DNA replication by interacting with the FtsZ protein which is essential for cell division.     

Comparison of the regional distribution of calspectin (nonerythroid spectrin or fodrin), alpha-actinin, vinculin nonerythroid protein 4.1, and calpactin in normal and avian sarcoma virus- or Rous sarcoma virus-induced transformed cells

With fluorescence and interference reflection microscopy (IRM), we compared the regional distribution of calspectin, its interacting proteins (nonerythroid protein 4.1 and calpactin), alpha-actinin, and vinculin in NRK cells and their avian sarcoma virus (ASV)- or temperature-sensitive (ts) Rous sarcoma virus (RSV)-transformed cells. The localization of these cytoskeletal proteins was determined with the specific antibodies. In NRK cells, alpha-actinin and vinculin were concentrated at adhesion plaques. By contrast, calspectin was distributed throughout the cytoplasm, but not concentrated at adhesion plaques. In ASV- and ts RSV-transformed cells, all three cytoskeletal proteins were concentrated at dot structures representing cellular feet. Nonerythroid protein 4.1 and calpactin were diffusely distributed throughout the cytoplasm of NRK cells and their transformed counterparts. In the case of calpactin, a part of this protein was excluded near regions of the terminal ends of stress fibers. These two proteins did not show the restricted location at the dot structures of transformed cells. From these findings, it is apparent that the accumulation of calspectin into dot structures is a specific event for cell transformation induced by the src protein.     

Phospholipase C in human endometrial fibroblasts and its regulation by estrogens

1. Stimulated inositolphospholipid turnover has been proposed as a signal-transducing mechanism in many cell types. It appears to be initiated by stimulation of hydrolysis of inositolphospholipid by a phospholipase C. 2. In human endometrial fibroblasts, estradiol was observed to cause sequential enhancement of [32P]phosphate incorporation into phosphatidic acid (PA) and phosphatidylinositol (PI), indicating an accelerating effect of estradiol on inositolphospholipid turnover. Specific 32P-radioactivity in the gamma-phosphate of ATP was increased in response to estradiol. Estrone or estriol were without any effects. 3. To investigate possible mechanisms by which estradiol activates a phospholipase C enzyme in the fibroblasts, the plasma membrane fraction isolated from the fibroblasts was exposed to estradiol in the presence of guanosine triphosphate (GTP) to detect inositol trisphosphate (IP3) production. The IP3 production was Ca2+ dependent, a dependency not affected by estradiol. 4. However, ATP decreased the Ca2+ concentration required for IP3 production in a dose-dependent manner; adenosine diphosphate (ADP), cytidine triphosphate (CTP) showed no effects. 5. These findings from cell and cell-free systems might suggest that estradiol stimulates a phospholipase C, as a result of enhancement of intracellular ATP synthesis, but not as a result of a direct effect on the enzyme molecule or direct activation of receptor-phospholipase C unit. 6. This may give us new insight into estrogen-stimulated cellular phenomenon through some mechanisms other than that classically associated with the action of estrogen.