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.     

Selective diapedesis of Th1 cells induced by endothelial cell RANTES.

Differentiated CD4 T cells can be divided into Th1 and Th2 types based on the cytokines they produce. Differential expression of chemokine receptors on either the Th1-type or the Th2-type cell suggests that Th1-type and Th2-type cells differ not only in cytokine production but also in their migratory capacity. Stimulation of endothelial cells with IFN-gamma selectively enhanced transmigration of Th1-type cells, but not Th2-type cells, in a transendothelial migration assay. Enhanced transmigration of Th1-type cells was dependent on the chemokine RANTES produced by endothelial cells, as indicated by the findings that Ab neutralizing RANTES, or Ab to its receptor CCR5, inhibited transmigration. Neutralizing Ab to chemokines macrophage-inflammatory protein-1alpha or monocyte chemotactic protein-1 did not inhibit Th1 selective migration. Whereas anti-CD18 and anti-CD54 blocked basal levels of Th1-type cell adherence to endothelial cells and also inhibited transmigration, anti-RANTES blocked only transmigration, indicating that RANTES appeared to induce transmigration of adherent T cells. RANTES seemed to promote diapedesis of adherent Th1-type cells by augmenting pseudopod formation in conjunction with actin rearrangement by a pathway that was sensitive to the phosphoinositol 3-kinase inhibitor wortmannin and to the Rho GTP-binding protein inhibitor, epidermal cell differentiation inhibitor. Thus, enhancement of Th1-type selective migration appeared to be responsible for the diapedesis induced by interaction between CCR5 on Th1-type cells and RANTES produced by endothelial cells. Further evidence that CCR5 and RANTES play a modulatory role in Th1-type selective migration derives from the abrogation of this migration by anti-RANTES and anti-CCR5 Abs.     

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.     

Beta-sheet folding of fragment (16-36) of bovine pancreatic trypsin inhibitor as predicted by Monte Carlo simulated annealing.

A tertiary structure prediction is described using Monte Carlo simulated annealing for the peptide fragment corresponding to residues 16-36 of bovine pancreatic trypsin inhibitor (BPTI). The simulation starts with randomly chosen initial conformations and is performed without imposing experimental constraints using energy functions given for generic interatomic interactions. Out of 20 simulation trials, seven conformations show a sheet-like structure--two strands connected by a turn--although this sheet-like structure is not as rigid as that observed in native BPTI. It is also shown that these conformations are mostly looped and exhibit a native-like right-handed twist. Unlike the case with the C-peptide of RNase A, no conspicuous alpha-helical structure is found in any of the final conformations obtained in the simulation. However, the lowest-energy conformation does not resemble exactly the native structure. This indicates that the rigid beta-sheet conformation of native BPTI merely corresponds to a local minimum of the energy function if the fragment with residues 16-36 is isolated from the native protein. A statistical analysis of all 20 final conformations suggests that the tendency for the peptide segments to form extended beta-strands is strong for those with residues 18-24, and moderate for those with residues 30-35. The segment of residues 25-29 does not tend to form any definite structure. In native BPTI, the former segments are involved in the beta-sheet and the latter in the turn. A folding scenario is also speculated from this analysis.     

Life history of Japanese Stypocaulon durum (Sphacelariales, Phaeophyceae)

Phenology, morphology, life history and responses to different temperature and photoperiod conditions were studied in Japanese Stypocaulon durum (Ruprecht) Okamura. Erect thalli of the species were collected year-round, but the mature thalli forming either uniloc-ular sporangia or two different types of plurilocular structures (evidently gametangia) on separate thalli were found only in winter. ln culture, an isomorphic life history is suggested for the species, alternating between a sporophyte forming unilocular sporangia and gam-etophytes forming plurilocular macro- (female) and micro- (male) gametangia. Contents of unilocular sporangia were not released, but germinated in situ, developing into erect thalli forming plurilocular gametangia. Macrogametangia released aplanogametes (oospores), but male gametangia appeared to be non-functional, although flagellated cells were once formed in the loc-uli. This is the first report of plurilocular gametangia in the species. Although the species grew well and matured under considerably lower temperature conditions than European Stypocaulon scoparium (L.) Sauvageau, its temperature requirements showed similarity to northwestern Atlantic Stypocaulon species. This supports the notion that northwestern Atlantic Stypocaulon is conspecific with S. durum.     

Mechanism of How Salt-Gradient-Induced Charges Affect the Translocation of DNA Molecules through a Nanopore

Experiments using nanopores demonstrated that a salt gradient enhances the capture rate of DNA and reduces its translocation speed. These two effects can help to enable electrical DNA sequencing with nanopores. Here, we provide a quantitative theoretical evaluation that shows the positive net charges, which accumulate around the pore entrance due to the salt gradient, are responsible for the two observed effects: they reinforce the electric capture field, resulting in promoted molecule capture rate; and they induce cationic electroosmotic flow through the nanopore, thus significantly retarding the motion of the anionic DNA through the nanopore. Our multiphysical simulation results show that, during the polymer trapping stage, the former effect plays the major role, thus resulting in promoted DNA capture rate, while during the nanopore-penetrating stage the latter effect dominates and consequently reduces the DNA translocation speed significantly. Quantitative agreement with experimental results has been reached by further taking nanopore wall surface charges into account.     

Fruiting-inducing activity of cerebrosides observed with Schizophyllum commune

Isolation and identification of substances having an activity to stimulate the fruiting body formation of Schizophyllum commune were attempted. The active principles in its mycelia were divided into four fractions by sequential purification with silica gel column and reverse-phase HPLC column chromatography. By infrared spectra and thin-layer chromatography, the active substances in these four fractions were revealed as cerebrosides. About 0.1 μg of the cerebroside fractions showed a discriminative stimulating activity on S. commune when tested by the method these authors adopted. The active substance in the fraction II was N-2′-hydroxypalmitoyl-1-O-glucosyl-nonadecasphingadienine. The cerebrosides from pea seeds and Fusicoccum amygdali showed the similar activity on S. commune, but some commercial synthetic cerebrosides and cerebrosides from bovine and porcine brains exhibited no stimulating activity. Only definite cerebrosides with special structures seem to be able to induce the fruiting of S. commune.