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.     

Sequences of cDNAs for human salivary and pancreatic α-amylases

The nucleotide sequences of the cloned human salivary and pancreatic α-amylase cDNAs correspond to the continuous mRNA sequences of 1768 and 1566 nucleotides, respectively. These include all of the amino acid coding regions. Salivary cDNA contains 200 bp in the 5′-noncoding region and 32 in the 3′-noncoding region. Pancreatic cDNA contains 3 and 27 bp of 5′- and 3′-noncoding regions, respectively. The nucleotide sequence humology of the two cDNAs is 96% in the coding region, and the predicted amino acid sequences are 94% homologous.Comparison of the sequences of human α-amylase cDNAs with those previously obtained for mouse α-amylase genes (Hagenbuchle et al., 1980; Schibler et al., 1982) showed the possibility of gene conversion between the two genes of human α-amylase.     

Plant-growth regulators from common starfish (<Emphasis Type="Italic">Asterias amurensis</Emphasis> Lütken) waste

Starfish waste has been shown to be an effective compost material not only in the promotion of plant growth but also in terms of having insecticidal activity. In the present study, plant growth regulation by chemicals from starfish was examined. The aqueous fraction from a hot water extract of the starfish Asterias amurensis Lütken showed plant-growth activity, while the aqueous fraction from a methanol extract inhibited growth of Brassica campestris. The lipophilic fraction from the methanol extract also exhibited a plant growth-promoting effect. The active components from each extract were identified. Asterubine from the hot water extract promoted plant growth. A ceramide from the lipophilic fraction showed root growth promoting effect, and three glucocerebrosides had promotive effects on the entire plant. Asterosaponins were identified as the main growth inhibitors in the aqueous fraction of the methanol extract. These active compounds from starfish waste could be analyzed as potential plant growth regulators in agricultural applications in the future.     

Effects of fat content in the diet on hepatic peroxisomes of the rat

Effects of fat content in the diet on rat liver peroxisomes was examined. In the livers of rats fed for one week on the high-fat diet containing 30% fat, the cyanide-insensitive palmitoyl-CoA oxidation was accelerated to eight times that of control and the enzymic activities of catalase, carnitine acetyltransferase and carnitine palmitoyltransferase were elevated by the factors of 1.3, 5 and 2, respectively. In contrast, the activities of D-amino acid oxidase in addition to the three enzymes mentioned above were all lowered by 20% when the animals were maintained on a fat-free diet for the same period of time. It appears that the high-fat diet-induced increase in the activity of carnitine palmitoyltransferase is a result of the raised activity of this enzyme in mitochondria only while the apparent high activity reflects stimulation of carnitine acetyltransferase in all the subcellular fractions. Another notable effect of the high-fat diet was a remarkable increase in the quantity of a peroxisome-associated polypeptide which was separable by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It is noteworthy that this effect of the high-fat diet resemble that of clofibrate. If the diet was deprived of fat, however, this polypeptide species, with an estimated molecular weight of 80 000, decreased to a level slightly lower than normal. On the basis of the electron micrographic criteria, the high-fat diet provoked a marked proliferation of hepatic peroxisomes.     

Cd-Binding Complexes from the Root Tissues of Various Higher Plants Cultivated in Cd2+-Containing Medium

Stone parsley, soybean, sunflower, sweet potato, potato, andadlay cultivated in a Cd²⁺-containing medium had Cd-bindingcomplexes with molecular weights of about 4,000 in the roottissues. The complexes were similar to the complex previouslyfound in water hyacinth roots in their absorption and CD spectraand their amino acid compositions. The results indicate thewidespread existence of complexes similar to fission yeast Cd-BPlin roots of various plants. (Received June 30, 1986; Accepted December 18, 1986)     

Interaction between cytochrome c and cytochrome c peroxidase: excited-state reactions of zinc- and tin-substituted derivatives

The effect of cytochrome c peroxidase (CCP) and apoCCP on the fluorescence and phosphorescence of Zn and Sn cytochrome c (cyt c) and the effect of cyt c on the fluorescence and phosphorescence of Zn CCP were examined. We found the following: The fluorescence yields of Zn and Sn cyt c were quenched by about 20% by CCP, consistent with energy transfer between the two chromophores with a separation of about 1.8 nm. The phosphorescence spectrum of Zn cyt c (but not Sn cyt c) shifts by 20 nm to the blue upon complexation with either CCP or apoCCP; at the same time the phosphorescence lifetime of Zn cyt c decreases from 12 +/- 2 to 6 ms with apoCCP addition. Zn CCP phosphorescence decay increases from 8.3 to 9.1 ms upon addition of poly(L-lysine) used to mimic cyt c. It is concluded from these results that binding of the redox partner or an analogue to Zn CCP and Zn cyt c results in a conformational change. The respective phosphorescence lifetimes of Zn and Sn cyt c were 13 and 3 ms in the absence of CCP and 1.6 and 1.1 ms in the presence of CCP; this corresponds to a quenching rate due to CCP of 519 and 570 s-1, for Zn and Sn cyt c, respectively. The phosphorescence of Zn CCP is also affected by native cyt c but is dramatically less than the complementary pair; the quenching rate constant is 17 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Participation of peroxisomes in lipid biosynthesis in the harderian gland of guinea pig.

Peroxisomal enzyme activities in the guinea-pig harderian gland, which has a unique lipid composition, were studied. Activities of catalase, acyl-CoA oxidase and the cyanide-insensitive acyl-CoA beta-oxidation system in this tissue were comparable with those in rat liver. The activities of dihydroxyacetone phosphate acyltransferase (DHAPAT, EC 2.3.1.42) and alkyl-DHAP synthase (EC 2.5.1.26) were appreciable, and the distributions of both activities were consistent with that of sedimentable catalase activity. Glycerol-3-phosphate acyltransferase (GPAT, EC 2.3.1.15), which is localized in both microsomes (microsomal fractions) and mitochondria in the rat liver, was a peroxisomal enzyme in the harderian gland, though the activity was only about one-tenth of the DHAPAT activity. These enzymes had different pH profiles and substrate specificity. The existence of high activities of enzymes of the acyl-DHAP pathway in peroxisomes suggests the physiological significance of peroxisomes in the biosynthesis of glycerol ether phospholipid and 1-alkyl-2,3-diacylglycerol in the guinea-pig harderian gland.     

Lung burden of green nickel oxide aerosol and histopathological findings in rats after continuous inhalation

There are few inhalation studies of nickel carcinogenesis. In this study, Wistar male rats were exposed to green nickel oxide (NiO(G)) aerosols (mass median aerodynamic diameter, 0.6 μm) for 7 h/d, 5 d/wk for up to 12 mo. The average exposure concentration was controlled at 0.3 and 1.2 mg/m³ during the exposure. For histopathological examination and measurement of the nickel concentration in rat organs, the rats were sacrificed at 3, 6, and 12 mo of exposure and 8 mo clearance period following 12 mo of exposure. The nickel content in rat lungs that was observed up to 2.6 mg after 12 mo exposure, was proportional to the exposure concentration during the exposure. The clearance of the nickel from the lungs was very slow and the biological half time was determined 7.7 mo. Although the rats were exposed continuously to NiO(G), for 12 mo and kept for 8 mo clearance period, there were no malignant tumors in any of the exposed animals.