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.     

Fractal property of eye movements in schizophrenia

 On the basis of a temporal model of animal behavior we conducted temporal analysis of eye movements in schizophrenic subjects (n=10) and normal controls (n=10). We found a fractal property in schizophrenic subjects, the fixation time of eye movement during reading ambiguous and difficult sentences showing a clear inverse power law distribution. An exponential distribution of a nonfractal nature was found in normal controls. Received: 21 July 1995/Accepted in revised form: 30 April 1996     

Conformation of NAD+ bound to allosteric L-lactate dehydrogenase activated by chemical modification

On modification of arginine residues with 2,3-butanedione, the Thermus caldophilus L-lactate dehydrogenase is converted to an activated form that is independent of an allosteric effector, fructose 1,6-bisphosphate (Fru-1,6-P2). The conformation of NAD+ bound to the modified enzyme in the absence of Fru-1,6-P2 was investigated by means of proton NMR, analyzing the time dependence of the transferred nuclear Overhauser effect (TRNOE) and TRNOE action spectra. The inter-proton distances determined on TRNOE analysis indicated that both the nicotinamide riboside moiety and the adenosine moiety of NAD+ were in the anti conformation, the ribose rings being in the C3'-endo form. This conformation was almost the same as that of NAD+ bound to the native enzyme-Fru-1,6-P2 complex, rather than that of NAD+ bound to the free native enzyme. These results suggest that the C3'-endo-anti form of the enzyme-bound NAD+ is essential for the activation of the T. caldophilus L-lactate dehydrogenase.     

Dermatan sulfate isomers in human articular cartilage characterized by high-performance liquid chromatography

The Constituents of dermatan sulfate isomers in human articular cartilage were studied at the disaccharide level by high-performance liquid chromatography. Appreciable amounts of dermatan sulfate components, i.e., dermatan sulfate, chondroitin sulfate types G and B, could be detected after digestion with chondroitinases-B or -ABC. The oversulfated dermatan sulfate isomers were isolated only after digestion with chondroitinase-ABC but not with the AC-lyase. The dermatan sulfate isomers were found to be markedly increased in weight loading parts of articular cartilage. It is postulated that the dermatan sulfate isomers are formed as a result of the weight loading reaction, which may be responsible for the fibrosis of articular cartilage.     

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)     

An ultrastructural study of HeLa cell invasion with Salmonella typhi GIFU 10007

Scanning electron micrograph of HeLa S3 monolayered cells, inoculated with viable bacteria of a Salmonella typhi strain GIFU 10007, revealed that the extended microvilli tangled the bacteria within 10 min after inoculation. The micrographs of HeLa cells, at 1 hr after inoculation, indicate the following: shortening of bacterium-attached microvilli, subsiding of tangled bacteria into microvilli bush, and then attachment of bacterial soma to cell surface making the cell membrane depressed. The transmission electron micrographs, at 1 hr after inoculation, demonstrated the findings of interaction between HeLa cell and S. typhi 10007, similar to those observed on scanning electron micrographs. Hair-like fine structures from the soma of challenge organisms were also observed. They were in contact with HeLa cell microvilli and cell membrane. The bacteria were first partially and then totally surrounded by the HeLa cell plasma membrane. One, two, or several bacteria with intact outer membrane were enclosed in intracytoplasmic membrane-bound vacuoles. Fragmented vacuolar membrane was still visible around the intracellularly accumulated bacteria at 24 hr after inoculation. The viable cells of S. typhi 10007 are regarded as internalizing into HeLa cells by a process of endocytosis and to multiply within the membrane-bound vacuoles.