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.     

Role of cell-mediated immunity in the resistance to experimental sporotrichosis in mice

The in vitro activity of several new imidazoles, cloconazole, sulconazole, butoconazole, isoconazole and fenticonazole, were compared with those of amphothericin B, flucytosine, and three azoles: econazole, miconazole and ketoconazole against isolates of pathogenic Candida. A total of 186 clinical isolates of 10 species of the genus Candida and two culture collection strains were tested by an agar-dilution technique. Isoconazole was the most active azole, followed by butoconazole and sulconazole. Differences between some of the species in their susceptibility to the antifungal agents were noted. Sulconazole and cloconazole had the highest activity in vitro against 106 isolates of C. albicans. Butoconazole and isoconazole were also very active against isolates of C. albicans, and were the most active azole compounds against 80 isolates of Candida spp.     

Clerodane diterpenoids from the roots of Portulaca pilosa

Three trans-clerodane diterpenoids, pilosanol A, B and C, the last compound being a glucoside, have been isolated from the roots of Portulaca pilosa. They show a marked contrast in skeletal type with the constituents of aerial part. Evolutionary changes in the biosynthetic abilities of Portulaca species is discussed.     

Formation of Chlorophyll-Protein Complexes in Greening Cucumber Cotyledons in Light and then in Darkness

The changes in chlorophyll-protein complexes (CPs) in cucumbercotyledons during illumination and subsequent dark incubationwere studied by SDS-polyacrylamide gel electrophoresis. Whenetiolated cucumber seedlings were illuminated, chlorophyll wassynthesized and CPs were formed. In the early phase of greening(6 h of illumination), light-harvesting chlorophyll a/b-proteincomplex (LHCP) was the main GP. As the greening proceeded, P700chlorophyll a-protein complex (CP1) accumulated. When 6-h illuminatedseedlings were transferred to darkness, CP1 accumulated concomitantlywith a decrease in LHCP without new chlorophyll synthesis. Thechanges in the amounts of CPs in the dark became smaller withthe progress of greening and were not observed after 72 h ofillumination. These changes were confirmed by examining thechlorophyll/P700 ratio and the low temperature absorption spectrumof cotyledons. These results suggest that in the early phaseof greening, CPs were unstable and their chlorophyll moleculeseasily exchanged with those of other kinds of CPs. (Received October 14, 1982; Accepted December 1, 1982)     

Phylogeography of the temperate grassland plant Tephroseris kirilowii (Asteraceae) inferred from multiplexed inter-simple sequence repeat genotyping by sequencing (MIG-seq) data

Journal of Plant Research - A group of temperate grassland plant species termed the “Mansen elements” occurs in Japan and is widely distributed in the grasslands of continental East...     

Initiation of the extrinsic pathway of blood coagulation: evidence for the tissue factor dependent autoactivation of human coagulation factor VII

Previous studies demonstrated proteolytic activation of human blood coagulation factor VII by an unidentified protease following complex formation with tissue factor expressed on the surface of a human bladder carcinoma cell line (J82). In the present study, an active-site mutant human factor VII cDNA (Ser344----Ala) has been constructed, subcloned, and expressed in baby hamster kidney cells. Mutant factor VII was purified to homogeneity in a single step from serum-free culture supernatants by immunoaffinity column chromatography. Mutant factor VII was fully carboxylated, possessed no apparent clotting activity, and was indistinguishable from plasma factor VII by SDS-PAGE. Cell binding studies indicated that mutant factor VII bound to J82 tissue factor with essentially the same affinity as plasma factor VII and was cleaved by factor Xa at the same rate as plasma factor VII. In contrast to radiolabeled single-chain plasma factor VII that was progressively converted to two-chain factor VIIa on J82 monolayers, mutant factor VII was not cleaved following complex formation with J82 tissue factor. Incubation of radiolabeled mutant factor VII with J82 cells in the presence of recombinant factor VIIa resulted in the time-dependent and tissue factor dependent conversion of single-chain mutant factor VII to two-chain mutant factor VIIa. Plasma levels of antithrombin III had no discernible effect on the factor VIIa catalyzed activation of factor VII on J82 cell-surface tissue factor but completely blocked this reaction catalyzed by factor Xa. These results are consistent with an autocatalytic mechanism of factor VII activation following complex formation with cell-surface tissue factor, which may play an important role in the initiation of extrinsic coagulation in normal hemostasis.     

Integrative genomic phylogeography reveals signs of mitonuclear incompatibility in a natural hybrid goby population

Hybridization between divergent lineages generates new allelic combinations. One mechanism that can hinder the formation of hybrid populations is mitonuclear incompatibility, that is, dysfunctional interactions between proteins encoded in the nuclear and mitochondrial genomes (mitogenomes) of diverged lineages. Theoretically, selective pressure due to mitonuclear incompatibility can affect genotypes in a hybrid population in which nuclear genomes and mitogenomes from divergent lineages admix. To directly and thoroughly observe this key process, we de novo sequenced the 747‐Mb genome of the coastal goby, Chaenogobius annularis, and investigated its integrative genomic phylogeographics using RNA‐sequencing, RAD‐sequencing, genome resequencing, whole mitogenome sequencing, amplicon sequencing, and small RNA‐sequencing. Chaenogobius annularis populations have been geographically separated into Pacific Ocean (PO) and Sea of Japan (SJ) lineages by past isolation events around the Japanese archipelago. Despite the divergence history and potential mitonuclear incompatibility between these lineages, the mitogenomes of the PO and SJ lineages have coexisted for generations in a hybrid population on the Sanriku Coast. Our analyses revealed accumulation of nonsynonymous substitutions in the PO‐lineage mitogenomes, including two convergent substitutions, as well as signals of mitochondrial lineage‐specific selection on mitochondria‐related nuclear genes. Finally, our data implied that a microRNA gene was involved in resolving mitonuclear incompatibility. Our integrative genomic phylogeographic approach revealed that mitonuclear incompatibility can affect genome evolution in a natural hybrid population.     

Effects of double rice cropping with irrigation on the diversity of herbaceous plants and their utilization as a food source in paddy fields of southern Lao People’s Democratic Republic

Herbaceous plant diversity including rare aquatic species has been lost in many countries by agricultural intensification and abandonment. In paddy fields of the Lao People’s Democratic Republic (Lao PDR), irrigation facilities have been constructed rapidly since 1997. The aim of this study was to clarify the impacts of double rice cropping accompanied by the introduction of irrigation systems on herbaceous plant diversity and utilization in paddy fields of southern Lao PDR. Ground vegetation surveys and interviews were conducted in Kok Deau and Lak 30 villages in Champasak Province, and propagule bank survey was conducted in Kok Deau village. The species richness and species diversity, measured by the Shannon’s diversity index, were not significantly different between the irrigated and rainfed paddies (p?>?0.05), when compared in both the wet and dry seasons. However, double rice cropping with irrigation systems affected herbaceous plant species composition in paddy fields. Increased use of chemical fertilizers in irrigated paddies resulted in predominance of tall undesirable species, such as Fimbristylis miliacea (L.) Vahl and Echinochloa colona (L.) Link. Small and frequently submerged species were dominant in the ground vegetation and propagule banks of the rainfed paddies. Since small submerged species are often sensitive to environmental changes, increase of irrigated paddy area may lead to a decrease in the variety of aquatic herbaceous plants in Lao PDR. While a total of 9 herbaceous plant species were utilized as foods in the villages, no change was recognized by farmers in species composition and frequency of utilization of paddy plants as food before and after the development of the irrigation systems.