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.     

Electron-microscopic and immunocytochemical analyses of Weibel-Palade bodies in the human umbilical vein during pregnancy

Summary The present study was done to elucidate the biological significance of the Weibel-Palade body of human umbilical vein endothelial cells. Quantitative determinations of these endothelial-specific granules throughout pregnancy revealed that their numbers and size per cell profile were maintained at low levels from 12 to 19 weeks of gestation; then both rapidly increased from 33 weeks to full term. This increase coincided with the development of the rough endoplasmic reticulum and an increase in the number of endothelial cell pinocytotic vesicles. Light-microscopic peroxidase anti-peroxidase and electron-microscopic protein A-gold techniques provided evidence that factor VIII-related antigen was localized in the Weibel-Palade bodies. Furthermore, in vitro treatment of incubated umbilical vein tissue with compound 48/80, a histamine releaser, induced degranulation of Weibel-Palade bodies from the endothelium. The present study indicates that Weibel-Palade bodies are storage sites of both histamine and factor VIII-related antigen and have an important role in the obliteration of this vessel.     

Rabbit autoantibodies to actin induced by immunization with modified homologous actins

Actin is a major antigen involved in the reaction of smooth muscle antibody positive sera from patients with chronic active hepatitis. In the present study, actin extracted from rabbit skeletal muscle was denatured by sodium dodecyl sulfate and was immunized into the rabbit, a homologous animal for actin. The rabbits, thus immunized, produced antibodies reactive with actins of homologous and heterologous animals. In addition, the antibodies showed reactivity with autologous actin. It indicates that the denatured homologous actin is capable of terminating immunological tolerance to actin and induces formation of autoantibody to rabbit actin. This phenomenon may be implicated in the occurrence of anti-actin antibody in sera from patients with chronic liver disease and several other diseases.     

Vertical variation in fecundity of the intertidal gastropodMonodonta labio caused by different growth rates between tidal zones

Monodonta labio (Gastropoda: Trochidae) occurs in a wide tidal zone on a boulder-covered shore in Amakusa, Japan. To investigate sources of variation in reproductive output within a population, the fecundity ofM. labio was estimated. Regressions of gonad egg counts on shell width were calculated from samples collected bi-weekly at three tidal zones: high, mid and low intertidal. Seasonal fluctuations in the regression revealed that individual females spawn at least three times a year. Variation in fecundity between the three tidal zones was not detected at any time in standard 12 mm snails. Due to the high growth rate in the low zone during the reproductive season, annual fecundity in the low zone was larger than that in the high and mid zones. Thus, tidal zone variation in fecundity ofM. labio was a result of growth variation between tidal zones.     

Modelling and analyzing density-dependent population processes: Comparison between wild and laboratory strains of the bean weevil,Callosobruchus chinensis (L.)

Three models were constructed for analyzing the population characteristics ofC. chinensis on stored beans; model A describing the whole reproductive process with a single equation, model B describing the three age-specific processes (oviposition, egg survival and larval survival) with separate equations, and model C which describes all these processes not for the whole habitat but for the individual beans comprizing it. The logit equation was employed here as a common basis to describe the density-response relationship involved. All three models showed very good fit to the experimental data obtained for both laboratory and wild strains of the weevil. The parameter values characterizing the population dynamics were, however, widely different between the two strains; the laboratory one which had been reared for some 500 generations showed significantly higher reproductive capacity, less sensitive and gentler response to crowding in both adult and egg stages, and more uniform egg distribution among individual beans, as compared with the wild strain newly introduced. Sensitivity analyses using these models suggested that these changes in population characteristics have been attained by the process of domestication or adaptation to stable laboratory conditions through a long period of time. This process seemed in effect to have optimized the population's performances in the laboratory environment. Evolutionary significance of such optimization was discussed with reference to the selection pressure which may have acted upon individuals.     

The effect of three novel thromboxane A2 receptor antagonists (S-1452, AA-2414 and ONO-3708) on the increase in pulmonary pressure caused by Forssman anaphylaxis in guinea-pigs.

The effects were studied of three novel thromboxane A2 (TXA2) receptor antagonists (S-1452, AA-2414 and ONO-3708) on the increase in pulmonary pressure caused by Forssman anaphylaxis in guinea-pigs. Three TXA2 antagonists at doses of between 1 and 10 mg/kg administered orally 1 h before the challenge clearly inhibited the pulmonary pressure increase. At a dose of 10 mg/kg, all three antagonists inhibited the pulmonary pressure increase caused by leukotriene D4 (LTD4) and U-46619, but not that caused by histamine. The decrease in peripheral platelet counts caused by Forssman anaphylaxis was also clearly inhibited by the three TXA2 antagonists. However, the decreased peripheral leukocyte counts were unaffected by the three agents. The decrease in serum complement activity (CH50) was inhibited by S-1452 and AA-2414 at a dose of 10 mg/kg. In bronchoalveolar lavage fluid (BALF), significant increases in eosinophils and neutrophils were observed after Forssman anaphylaxis. Three TXA2 antagonists at a dose of 10 mg/kg (except for AA-2414 on eosinophils) did not affect the changes of leukocyte counts in BALF. Moreover, increases in the TXB2 and 6-keto-PGF1 alpha levels of the BALF brought about by Forssman anaphylaxis were unaffected by the three TXA2 receptor antagonists. Histamine and LTD4 were not changed in the BALF after Forssman anaphylaxis. These results indicate the efficacy of TXA2 receptor antagonists on the increase in pulmonary pressure caused by Forssman anaphylaxis in guinea-pigs by direct antagonism to released TXA2.     

BAG-6 is essential for selective elimination of defective proteasomal substrates

BAG-6/Scythe/BAT3 is a ubiquitin-like protein that was originally reported to be the product of a novel gene located within the human major histocompatibility complex, although the mechanisms of its function remain largely obscure. Here, we demonstrate the involvement of BAG-6 in the degradation of a CL1 model defective protein substrate in mammalian cells. We show that BAG-6 is essential for not only model substrate degradation but also the ubiquitin-mediated metabolism of newly synthesized defective polypeptides. Furthermore, our in vivo and in vitro analysis shows that BAG-6 interacts physically with puromycin-labeled nascent chain polypeptides and regulates their proteasome-mediated degradation. Finally, we show that knockdown of BAG-6 results in the suppressed presentation of MHC class I on the cell surface, a procedure known to be affected by the efficiency of metabolism of defective ribosomal products. Therefore, we propose that BAG-6 is necessary for ubiquitin-mediated degradation of newly synthesized defective polypeptides.