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.     

Evidence of increased synthesis of δ-aminolevulinic acid dehydratase in experimental lead-poisoned rats

δ-Aminolevulinic acid dehydratase (porphobilinogen synthase; 5-aminolevulinate hydro-lyase, EC 4.2.1.24) was purified from rat and rabbit erythrocytes to a homogeneous state. Specific activities were 26.0 and 26.6 units/mg protein for the rat and rabbit enzymes, respectively, and their estimated molecular weight was 280 000, each consisting of 8 subunits of M_r 35 000. In order to quantitate rat δ-aminolevulinic acid dehydratase at several stages of lead-poisoning, a radioimmunoassay technique using goat antiserum against the rat enzyme was developed for the first time. This technique was specific, reproducible and high sensitive allowing determination of 1 ng enzyme. When drinking water containing 25 mM lead acetate was given daily to rats ad lib. the δ-aminolevulinic acid dehydratase activity in the blood, assayed without any pretreatment, decreased to 8% of the control level on the next day. On the contrary, the restored enzyme activity, assayed in the presence of Zn²⁺ and dithiothreitol, was greater than normal by the fourth day of lead administration in bone-marrow cells and by the ninth day in the peripheral blood. The increased activity level stayed the same from the ninth day onward. The enzyme content as determined directly by the radioimmunoassay technique at this stage was about 2-fold above that the control. There was no significant difference in the number of reticulocytes and the distribution profile of different types of reticulocytes between the lead-exposed and non-exposed rats. Therefore, the increase in the amount of δ-aminolevulinic acid dehydratase in erythrocytes of lead-poisoned rats was suggested to be due to an increased rate of synthesis in the bone-marrow cells.     

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.     

Cloning and nucleotide sequence of the isoamylase gene from Pseudomonas amyloderamosa SB-15

The gene (iam) coding for isoamylase (glycogen 6-glucanohydrolase) of Pseudomonas amyloderamosa SB-15 was cloned. Its nucleotide sequence contained an open reading frame of 2313 nucleotides (771 amino acids) encoding a precursor of secreted isoamylase. The precursor contained a signal peptide of 26 amino acid residues at its amino terminus and three regions homologous with those conserved in alpha-amylases (1,4-alpha-D-glucan 4-glucanohydrolase) of species ranging from prokaryotes to eukaryotes. These homologous regions were also found in another debranching enzyme, pullulanase (pullulan 6-glucanohydrolase) from Klebsiella aerogenes. Sequences of the isoamylase also showed significant homology with those between positions 300 and the carboxyl terminus of pullulanase. The regions required for the specificity of isoamylase were discussed on the basis of a comparison of its amino acid sequence with those of alpha-amylases, cyclomaltodextrin glucanotransferases, and pullulanase.     

Isolation and partial characterization of the major glycoproteins of horse and swine erythrocyte membranes

The major glycoproteins of horse and swine erythrocyte membranes were isolated and examined chemically and immunologically. The major glycoprotein of horse erythrocyte membranes had a molecular weight of 33 000 and consisted of 46.2% protein and 53.8% carbohydrate, of which 9.4% was hexose, 10.1% hexosamine and 33.7% sialic acid. This glycoprotein was associated with activity for the infectious mononucleosis heterophile antigen.There were two different major glycoproteins in swine erythrocyte membranes. One major glycoprotein had a molecular weight of 46 200 and consisted of 34.2% protein and 65.8% carbohydrate, of which 18% was hexose, 19% hexosamine and 27.2% sialic acid. This glycoprotein had phytohemagglutinin (Phaseolus vulgaris) binding activity. The other glycoprotein had a molecular weight of 29 000 and consisted of 50.4% protein and 49.6% carbohydrate, of which 6.4% was hexose, 7.0% hexosamine and 36.3% sialic acid. This glycoprotein had weak or absent phytohemagglutinin binding activity.     

Association between light exposure at night and manic symptoms in bipolar disorder: cross-sectional analysis of the APPLE cohort

ABSTRACT

Previous studies have found that keeping the room dark at night was associated with a decrease in manic symptoms for patients with bipolar disorder (BD). However, the association between light at night of real-life conditions and manic symptoms is unclear. We investigated the association between bedroom light exposure at night and manic symptoms in BD patients. One-hundred and eighty-four outpatients with BD participated in this cross-sectional study. The average light intensity at night during sleep was evaluated using a portable photometer for seven consecutive nights. Manic symptoms were assessed using the Young Mania Rating Scale (YMRS), and scores ≥5 were treated as a “hypomanic state.” The median (interquartile range) YMRS score was 2.0 (0–5.0), and 52 (28.2%) participants were in a hypomanic state. The prevalence of a hypomanic state was significantly higher in the participants with an average light intensity at night exposure of ≥3 lux than in those with <3 lux (36.7% versus 21.9%; P = .02). In multivariable logistic regression analysis adjusted for BD type, depressive symptoms, sleep duration, and daytime physical activity, the odds ratio (OR) for a hypomanic state was significantly higher for the participants with an average light intensity at night exposure of ≥3 lux than for those with <3 lux (OR: 2.15, 95% confidence interval: 1.09–4.22, P = .02). This association remained significant at the cutoff value of YMRS score ≥6 (OR: 2.51, 95% confidence interval: 1.15–5.46; P = .02). The findings of this study indicate bedroom light exposure at night is significantly associated with manic symptoms in BD patients. Although the results of this cross-sectional investigation do not necessarily imply causality, they may serve to inform beneficial nonpharmacological intervention and personalized treatment of BD patients.     

Effects of Protamine Sulfate on Dengue Type 1 Viral Activities

Protamine treatment of type 1 dengue-infected mouse brain suspension resulted in precipitation of several viral specific activities. Complement-fixation activity was almost completely precipitated by protamine. The complement-fixation components recovered in the precipitate were comparable to a non-precipitated reference dengue 1 antigen in their homologous and heterologous reactions. Dengue hemagglutinin was also precipitated by the same treatment. The precipitated hemagglutinin was resolved into three components by buoyant density centrifugation, whose densities were 1.236, 1.215, and 1.178 g/ml, respectively. Three similar HA components were detected in non-protamine treated virus preparations. In both instances the highest-titered HA fraction possessed a buoyant density of 1.21–1.22 g/ml. These HA components were tested in the hemagglutination-inhibition reaction and were proved to be virus-specific. Cesium chloride density gradient centrifugation was shown to be useful for removing possible inhibitor(s) of viral specific hemagglutinin.     

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).