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.     

Physico-chemical control of the growth of a diatom, Asterionella formosa Hass., in a shallow eutrophic lake

Physico-chemical environmental control of the growth rate ofan algal population, the pennate diatom Asterionella formasaHass., was investigated for a year in natural water. This speciesoccurred in vegetative form in the water column all the time,although its population density varied by five orders of magnitudefrom a maximum of 1.4?10⁶ cells l^–1 in February to a minimumof 0.8?10¹ cells l^–1 in August. A mathematical model ofa combination of multiplicative and Liebig types suggested thatvegetative cells were severely limited in their growth rate,reaching almost 80% depression of the maximum rate between Decemberand February by low temperatures and nearly 50% depression inwarm seasons by light intensity. Nutrient limitations were onlyobserved in May, August and September, although they were notas great as by temperature and light intensity in the studylake. Population changing rates of A.formosa determined in thelake were low and agreed well with the estimated growth ratesduring winter, but those in summer were low and disagreed withthe estimated high growth rates. This suggests that populationchange of the species was highly dependent upon the specificgrowth rate in winter, but other factors became predominantin summer.     

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.     

An Application of Wastewater Treatment in a Cold Environment and Stable Lipase Production of Antarctic Basidiomycetous Yeast Mrakia blollopis

Milk fat curdle in sewage is one of the refractory materials for active sludge treatment under low temperature conditions. For the purpose of solving this problem by using a bio-remediation agent, we screened Antarctic yeasts and isolated SK-4 strain from algal mat of sediments of Naga-ike, a lake in Skarvsnes, East Antarctica. The yeast strain showed high nucleotide sequence homologies (>99.6%) to Mrakia blollopis CBS8921^T in ITS and D1/D2 sequences and had two unique characteristics when applied on an active sludge; i.e., it showed a potential to use various carbon sources and to grow under vitamin-free conditions. Indeed, it showed a biochemical oxygen demand (BOD) removal rate that was 1.25-fold higher than that of the control. We considered that the improved BOD removal rate by applying SK-4 strain was based on its lipase activity and characteristics. Finally, we purified the lipase from SK-4 and found that the enzyme was quite stable under wide ranges of temperatures and pH, even in the presence of various metal ions and organic solvents. SK-4, therefore, is a promising bio-remediation agent for cleaning up unwanted milk fat curdles from dairy milk wastewater under low temperature conditions.     

Differential expression of VGLUT3 in laboratory mouse strains: Impact on drug‐induced hyperlocomotion and anxiety‐related behaviors

The atypical vesicular glutamate transporter VGLUT3 is present in subpopulations of GABAergic interneurons in the cortex and the hippocampus, in subgroups of serotoninergic neurons in raphe nuclei, and in cholinergic interneurons in the striatum. C56BL/6N mice that no longer express VGLUT3 (VGLUT3^?/?) display anxiety‐associated phenotype, increased spontaneous and cocaine‐induced locomotor activity and decreased haloperidol‐induced catalepsy. Inbred mouse strains differ markedly in their sensitivity to anxiety and behavioral responses elicited by drugs. The purpose of this study was to investigate strain differences in VGLUT3 expression levels and its potential correlates with anxiety and reward‐guided behaviors. Five inbred mouse lines were chosen according to their contrasted anxiety and drugs sensitivity: C57BL/6N, C3H/HeN, DBA/2J, 129/Sv, and BALB/c. VGLUT3 protein expression was measured in different brain areas involved in reward or mood regulation (such as the striatum, the hippocampus, and raphe nuclei) and genetic variations in Slc17a8, the gene encoding for VGLUT3, have been explored. These five inbred mouse strains express very different levels of VGLUT3, which cannot be attributed to the genetic variation of the Slc17a8 locus. Furthermore, mice behavior in the open field, elevated plus maze, spontaneous‐ and cocaine‐induced locomotor was highly heterogeneous and only partially correlated to VGLUT3 levels. These data highlight the fact that one single gene polymorphism could not account for VGLUT3 expression variations, and that region specific VGLUT3 expression level variations might play a key role in the modulation of discrete behaviors.     

Autoregulation of Osteocyte Sema3A Orchestrates Estrogen Action and Counteracts Bone Aging

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Age-Associated Different Transcriptome Profiling in Zebrafish and Rats: an Insight into the Diversity of Vertebrate Aging

Marine Biotechnology - Most mammals, including humans, show obvious aging phenotypes, for example, loss of tissue plasticity and sarcopenia. In this regard, fish can be attractive models to study...     

Structural Analysis of a Trimer of β2-Microgloblin Fragment by Molecular Dynamics Simulations

A peptide ${\beta }_2$-${\text{m}}_{21?31}$, which is a fragment from residue 21 to residue 31 of ${\beta }_2$-microgloblin, is experimentally known to self-assemble and form amyloid fibrils. In order to understand the mechanism of amyloid fibril formations, we applied the replica-exchange molecular dynamics method to the system consisting of three fragments of ${\beta }_2$-${\text{m}}_{21?31}$. From the analyses on the temperature dependence, we found that there is a clear phase transition temperature in which the peptides aggregate with each other. Moreover, we found by the free energy analyses that there are two major stable states: One of them is like amyloid fibrils and the other is amorphous aggregates.