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.     

High resolution nuclear magnetic resonance study of base pairing in four purified transfer RNA molecules

The high resolution nuclear magnetic resonance spectra of hydrogen bonded protons in four purified tRNA molecules are reported. From the temperature and concentration dependence it is shown that these resonances arise from intramolecular hydrogen bonds.     

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.     

NESTMATE RECOGNITION AND KIN RECOGNITION IN ANTS

Abstract  All ants (Hymenoptera, Formicidae) are highly eusocial insects that are characterized by reproductive division of labor with sterile castes (worker and soldier) helping fertile castes (queen and male) to reproduce.
Ant societies, like other complex animal societies, have developed a sophisticated communication system, in which recognition behaviors are frequently involved Recognition abilities allow individuals to orient and modulate their behaviors effectively and appropriately in response to the characteristics andlor signals expressed by other organisms. Among recognition behaviors, nestmate recognition and kin recognition mechanisms have attracted great attention of sociobiologists, ecologists, insect physiologists and biochemists since 1970's. This is parallel with the popularization of Hamilton's kin selection theory. The present paper aims at reviewing the current understanding on nestmate/kin recognition in ants. This review consists of three parts. The first part concerns the diversity of recognition behaviors and their ecological implications with emphasis on nestmatelkin recognition; in the second part, the current understandings on the mechanism of nestmatelkin recognition are outlined; and in the third part, we discuss the ontogenetic development of nestmate recognition behavior and naturally mixed colonies. The study of the integration mechanism of social parasite may provide heuristic clues to the understanding of kin/nestmate recognition system.     

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).     

Roles of neonatal and prepubertal testicular androgens on androgen-induced proliferative response of seminal vesicle cells in adult mice

Male mice were castrated at 0, 10, 20, 30, 40 and 60 days of age; daily injections of testosterone propionate (TP, 4 micrograms/g b. wt) were started from day 90. On various days after starting the TP injections, the incorporation of 5-[125I]iodo-2'-deoxyuridine into the whole seminal vesicles was determined as an index for proliferation. The seminal vesicle cells in mice castrated on days 0 and 20 were characterized by low weight (0.5-1 mg) before TP injection, long duration of androgen-induced proliferation (greater than 20 days) with a low peak, and involvement of both epithelial and fibromuscular cells (neonatal castration type). The seminal vesicle cells in mice castrated on days 60 and 40 were characterized by relatively high weight (5-10 mg) before TP injection, short duration of androgen-induced proliferation (10 days) with a high peak, and involvement of only the epithelial cells (adult castration type). In mice castrated on days 0 and 20, the neonatal castration type of androgen-induced proliferation was completely changed to the adult castration type when TP pretreatment (2 micrograms/g b. wt per 12 h) had been given from day 20 to day 40. However, the TP pretreatment given from day 90 to day 110 instead of days 20-40 had no such effect in 140-day old mice castrated on day 0. The present findings suggest that testicular androgens secreted from day 20 to day 40 play an indispensable role in the induction of irreversible proliferative response of the mouse seminal vesicle. The activity of the prepubertal androgens may not be completely compensated by androgen activity at adulthood.     

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)     

In vitro translocation of protein across Escherichia coli membrane vesicles requires both the proton motive force and ATP

The energy requirement for protein translocation across membrane was studied with inverted membrane vesicles from an Escherichia coli strain that lacks all components of F1F0-ATPase. An ompF-lpp chimeric protein was used as a model secretory protein. Translocation of the chimeric protein into membrane vesicles was totally inhibited in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP) or valinomycin and nigericin and partially inhibited when either valinomycin or nigericin alone was added. Depletion of ATP with glucose and hexokinase resulted in the complete inhibition of the translocation process, and the inhibition was suppressed by the addition of ATP-generating systems such as phosphoenolpyruvate-pyruvate kinase or creatine phosphate-creatine kinase. These results indicate that both the proton motive force and ATP are required for the translocation process. The results further suggest that both the membrane potential and the chemical gradient of protons (delta pH), of which the proton motive force is composed, participate in the translocation process.