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.     

Predator detection and avoidance by starlings under differing scenarios of predation risk

Practically all animals must find food while avoiding predators.An individual's perception of predation risk may depend on manyfactors, such as distance to refuge and group size, but it isunclear whether individuals respond to different factors ina similar manner. We tested whether flocks of foraging starlingsresponded in the same way to an increased perception of predationrisk by assessing three factors: (1) neighbor distances, (2)habitat obstruction, and (3) recent exposure to a predator.We found that in all three scenarios of increased risk, starlingsreduced their interscan intervals (food-searching bouts), whichincreased the frequency of their vigilance periods. We thenexamined how one of these factors, habitat obstruction, affectedescape speed by simulating an attack with a model predator.Starlings were slower to respond in visually obstructed habitats(long grass swards) and slower when they had their head downin obstructed habitats than when they had their head down inopen habitats. In addition, reaction times were quicker whenstarlings could employ their peripheral fields of vision. Ourresults demonstrate that different sources of increased riskcan generate similar behavioral responses within a species.The degree of visibility in the physical and social environmentaffects both the actual and perceived risk of predation.     

An Arabidopsis lipid map reveals differences between tissues and dynamic changes throughout development

Mass spectrometry is the predominant analytical tool used in the field of plant lipidomics. However, there are many challenges associated with the mass spectrometric detection and identification of lipids because of the highly complex nature of plant lipids. Studies into lipid biosynthetic pathways, gene functions in lipid metabolism, lipid changes during plant growth and development, and the holistic examination of the role of plant lipids in environmental stress responses are often hindered. Here, we leveraged a robust pipeline that we previously established to extract and analyze lipid profiles of different tissues and developmental stages from the model plant Arabidopsis thaliana. We analyzed seven tissues at several different developmental stages and identified more than 200 lipids from each tissue analyzed. The data were used to create a web-accessible in silico lipid map that has been integrated into an electronic Fluorescent Pictograph (eFP) browser. This in silico library of Arabidopsis lipids allows the visualization and exploration of the distribution and changes of lipid levels across selected developmental stages. Furthermore, it provides information on the characteristic fragments of lipids and adducts observed in the mass spectrometer and their retention times, which can be used for lipid identification. The Arabidopsis tissue lipid map can be accessed at http://bar.utoronto.ca/efp_arabidopsis_lipid/cgi-bin/efpWeb.cgi .     

A comparison of ferric-chelate reductase and chlorophyll and growth ratios as indices of selection of quince,pear and olive genotypes under iron deficiency stress

Quince (Cydonia oblonga Mill.), pear (Pyrus communis L.) and olive (Olea europaea L.) genotypes were evaluated for their tolerance to iron deficiency stress by growing young plants in three types of aerated nutrient solutions: (1) with iron, (2) without iron or (3) low in iron and with 10 mM bicarbonate. Plants were obtained either from rooted softwood cuttings or from germination of seeds. The degree of tolerance was evaluated with several indices: (1) the chlorophyll content, (2) the root Fe³⁺ reducing capacity and (3) the whole plant relative growth. Fifteen hours before Fe³⁺ reducing capacity determination, iron was applied to the roots of plants with iron-stress, since this method resulted in increasing the reductase activity. All quince and pear genotypes increased the root Fe³⁺ reducing capacity when grown in the treatments for iron-stress, in relation to control plants of the same genotypes. In olive cultivars, the Fe³⁺ reducing capacity was lower in the iron-stress treatments than in the control one. Studying the relationship between relative growth and chlorophyll content for each genotype under iron-stress, in relation to both indices in control plants, a classification of species and genotypes was established. According to that, most olive cultivars and some pear rootstocks and cultivars appear more iron-efficient than quince rootstocks. Our study shows that in some woody species, determining root Fe³⁺ reducing capacity is not the best method to establish tolerance to iron deficiency stress.     

A New Non-Mendelian Genetic Element of Yeast That Increases Cytopathology Produced by M1 Double-Stranded RNA in ski Strains

The Saccharomyces cerevisiae SKI (superkiller) genes are repressors of replication of M, L-A, and L-BC double-stranded (ds) RNAs; ski strains have an increased M dsRNA copy number and, as a result, are cold-sensitive for growth at 8 degrees. Growth is normal, however, at higher temperatures. We have found a new cytoplasmic genetic element [D] (for disease) that makes M1 dsRNA-containing superkiller strains grow slowly at 30 degrees, not at all at 37 degrees, and only very poorly at 20 degrees. These growth defects require three factors: a chromosomal ski mutation, the presence of M1 dsRNA, and the presence of the new cytoplasmic factor, [D]. We have isolated mutants unable to maintain [D] (mad), at least one of which is due to mutation of a single chromosomal locus. Further, [D] can be cured by growth at 37-39 degrees. We present evidence that [D] is not M, L-A, L-BC or W dsRNAs or mitochondrial DNA, 2 mu DNA, or [psi], but [D] depends on L-A for its maintenance. We also show that [D] is distinct from [B], a cytoplasmic element that allows M1 dsRNA to be stably replicated and maintained in spite of defects in certain chromosomal MAK genes that would otherwise be necessary. [D] activity is blocked by the presence of another extrachromosomal element, called [DIN] (for [D] interference). [D] and [DIN] may be different natural variants of the same molecule.     

Fatty acids bound to vitamin D-binding protein (DBP) from human and bovine sera

Human and bovine vitamin D-binding protein (DBP) have been isolated from serum by a method that does not involve denaturing steps. This method includes Cibacron Blue-Sepharose chromatography, gel filtration, DEAE-Sephadex chromatography and albumin immunoadsorption. Analysis of fatty acids bound to the isolated human and bovine DBP showed molar ratios of fatty acid to protein of 0.4 and 1.3 respectively meanwhile human and bovine albumin have bound 1.8 and 1.5 moles per mol respectively. Most of fatty acids bound to human and bovine DBP are monounsaturated and saturated, mainly oleic and palmitic acids, which together account for 50% of the total of fatty acids in both species. By contrast, polyunsaturated fatty acids represented a minor component, less than 5%.     

Analysis of pyridine dinucleotides in cultured rat hepatocytes by high-performance liquid chromatography

An isocratic reverse-phase high-performance liquid chromatography method for the separation and quantitation of total pyridine dinucleotides in hepatocyte cultures is described. Cells are extracted with cold 3 M perchloric acid or 0.5 N sodium hydroxide containing 50% (v/v) ethanol and 35% cesium chloride for the determination of the oxidized or reduced pyridine dinucleotides, respectively. Pyridine dinucleotides in the neutralized extracts were separated on an Excellopak ODS C18 (4.6 X 150 mm) column with 0.1 M potassium phosphate, pH 6.0, containing 3.75% methanol as the mobile phase. NAD+ and NADP+ were detected spectrophotometrically at 254 nm. The response was linear from 5 to 4000 pmol with recoveries of NAD+ and NADP+ of 98 and 101.1%, respectively. NADH and NADPH were monitored fluorometrically by activation at 370 nm and emission in the 400-700 nm range. The reduced pyridine dinucleotides had a linear response from 7.5 to 60 pmol with recoveries of NADH and NADPH of 99.4 and 101.3%, respectively. The coefficients of variation for all of the pyridine dinucleotide standards were less than 3.5%.     

The caudal spinal cord of coho salmon (Oncorhynchus kisutch): Immunocytochemical evidence of a “caudal serotoninergic system”

Summary The caudal spinal cord of the coho salmon was investigated by means of immunocytochemistry using antisera against serotonin, urotensin I, urotensin II, somatostatin and a urea-extract of bovine Reissner's fiber (AFRU). Populations of serotonin-immunoreactive (IR) neurons were found rostral and dorsal to the urophysis in close spatial association with caudal secretory neurons. Thick, smooth serotonin-IR processes extended toward the external surface of the spinal cord where they displayed conspicuous terminal dilatations. Thin, beaded serotonin-IR fibers appeared to innervate populations of caudal secretory and somatostatin-IR cerebrospinal fluid-contacting neurons. Most caudal neurosecretory cells displayed both urotensin I and urotensin II immunoreactivities; only a minority reacted exclusively with either urotensin I or urotensin II antisera. Urotensin II-IR and somatostatin-IR cerebrospinal fluid (CSF)-contacting neurons were found as an integral component of the central canal wall in the caudal spinal cord and filum terminale; their dendritic processes appeared to contact Reissner's fiber, which displayed a weak AFRU-immunoreactivity while inside the central canal, but became strongly reactive in the interior of the terminal ventricle as it formed the massa caudalis. The distribution of serotoninergic processes points to a regulatory role in the function of caudal secretory and CSF-contacting neurons and to a putative serotonin release into the subarachnoid space and/or meningeal vasculature. It is also suggested that the CSF-contacting neurons of the central canal may participate in a feedback mechanism controlling the secretory activity of the subcommissural organ.Supported by Grant A/1095-1 from the International Foundation for Science, Sweden, to C.Y.; Grant I/63-476 from Volkswagen-Stiftung to E.R.; and Grant S-85-39 from the Dirección de Investigaciones, Universidad Austral de Chile     

Anatomy of the hominoid wrist joint: Its evolutionary and functional implications

Observations on the behavior of living hominoids show generic differences in the use and posture of the wrist joint. Both orang-utans and hylobatids usually use the wrist in suspensory behaviors. However, orang-utans emphasize markedly adducted and flexed wrist postures, while hylobatids emphasize violent forearm and wrist rotation. African apes, especially the gorilla, use the wrist more frequently than other hominoids for terrestrial quadrupedal weight-bearing. Humans use the wrist less frequently for supportive purposes than do other hominoids. These behavioral differences correspond to structural specializations in the proximal carpal joint of each of the hominoid genera. Although each of the hominoid genera has apparently modified its proximal carpal joint best to serve its characteristic behaviors, all hominoids share a unique proximal carpal joint that permits approximately 160ℴ of forearm rotation. The hylobatid proximal carpal joint is specialized in exhibiting a marked development of those structures limiting forearm rotation, but it is in most respects the least derived— that is, closest to the nonhominoid anthropoids. Chimpanzees show a proximal carpal joint that is more generalized than those of the other great apes but more derived than that of hylobatids. The human and gorilla proximal wrist joints, on the other hand, show marked modifications for weight-bearing in terrestrial behaviors. Orang-utans have the most derived proximal carpal joint, which in many respects parallels that of the slow-climbing nonhominoid primates. The comparative anatomy and structural specializations of the wrist joint support (a) an early divergence of hylobatids from the common hominoid stock, (b) a common ancestry for gorillas and humans separate from the other hominoids, and (c) a long independent evolutionary period for orang-utans since their divergence from the common hominoid stock, or one that was marked by strong selection pressures for wrist specializations. Unfortunately, the generalized condition of the chimpanzee’s wrist joint and the very derived condition of the orang-utan wrist provide uncertain evidence as to which of the two was first to diverge from the common hominoid stock. Identification of hominoid wrist specializations as reflecting real phylogenetic relationships or parallelisms depends on how well the phytogeny inferred from wrist morphology accords with those arrived at from the study of other systems.