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.     

Cancer chemotherapy and somatic cell mutation

The occurrence of a second neoplasm is one of the major obstacles in cancer chemotherapy. The elucidation of the genotoxic effects induced by anti-cancer drugs is considered to be helpful in identifying the degree of cancer risk. Numerous investigations on cancer patients after chemotherapy have demonstrated: (i) an increase in the in vivo somatic cell mutant frequency (Mf) at three genetic loci, including hypoxanthine–guanine phosphoribosyl-transferase (hprt), glycophorin A (GPA), and the T-cell receptor (TCR), and (ii) alterations in the mutational spectra of hprt mutants. However, the time required for and the degree of such changes are quite variable among patients even if they have received the same chemotherapy, suggesting the existence of underlying genetic factor(s). Accordingly, some cancer patients prior to chemotherapy as well as patients with cancer-prone syndrome have been found to show an elevated Mf. Based on the information obtained from somatic cell mutation assays, an individualized chemotherapy should be considered in order to minimize the risk of a second neoplasm.     

Effect of IL-2 in vitro on CTL generation in spleen cells of young and old mice: asialo GM1+ cells are required for the apparent restoration of the CTL response

The role of asialo GM1+ (ASGM1+) cells and exogenous IL-2 in the age-related decline in allospecific CTL activity was evaluated. Primary CTL were generated in mixed leukocyte culture (MLC) [BALB/cANN (H-2d) anti C57BL/6N (H-2b)] and tested for allospecific lytic activity against the EL-4 (H-2b) cell culture line, and for non-MHC-restricted activity against WEHI-3 (H-2d) and YAC-1 (H-2a). Cultures included responder cell populations which had been treated with antibody to ASGM1 plus complement or complement alone, and irradiated stimulator cells, in the presence or absence of rIL-2 or crude IL-2-containing supernatants. The amount of rIL-2 used to accommodate the age-related decline in IL-2 production was determined empirically to be 500 U by assessing IL-2 production in MLCs containing responder cells from young versus old animals. rIL-2 appeared to restore the allospecific CTL activity generated by spleen cells of old mice to the level of that of young. However, treatment with anti-ASGM1 antibody revealed that this restoration was due to an effect of the IL-2 on ASGM1+ cells. The allospecific target cells, EL-4, were not sensitive to lymphokine-activated killer (LAK) cells induced by IL-2 alone under the conditions used. It is suggested that the apparent restoration was due to increased LAK-like (or MHC-nonrestricted) activity mediated by an ASGM1+ cell in the CTL precursor population.     

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.     

Three types of vitronectin in human blood

Vitronectin is a cell-adhesive glycoprotein in serum and plasma, also termed serum spreading factor and complement S-protein. It consists of a mixture of a polypeptide of molecular weight 75 kilodalton (kDa) and its nicked product of 65 kDa plus 10 kDa. By a quantitative immunoblotting assay, human blood samples could be classified into three distinct vitronectin types; type I (58% of the population) was 75 kDa rich and 65 kDa poor, type II (35% of the population) contained approximately equal amounts of 75 kDa and 65 kDa, and type III (5% of the population) was 75 kDa poor and 65 kDa rich. The vitronectin type did not correlate with age, sex, or ABO blood type.     

Biosynthesis of Purine Alkaloids in Camellia Plants

The metabolism of [8-¹⁴C]adenine and [8-¹⁴C]hypoxanthine infour species of Camellia plants was investigated in relationto the synthesis of purine alkaloids, caffeine and theobromine.Young leaves of C. sinensis had the ability to synthesize caffeine,but in C. irrawadiensis, these labelled precursors were incorporatedinto theobromine, not caffeine. No synthesis of purine alkaloidscould be detected in C. japonica and C. sasanqua leaves. Conventional"salvage" and degradation pathways of purines were present inall species of Camellia plants examined. ¹ Present address: Research Center, Mitsubishi Chemical IndustriesLtd., 1000 Kamisida-cho, Midori-ku, Yokohama, 227 Japan. (Received September 29, 1986; Accepted January 22, 1987)     

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)     

Reactive sulfhydryl groups of sarcoplasmic reticulum ATPase. I. Location of a group which is most reactive with N-ethylmaleimide

Ca2+-Transporting ATPase of rabbit skeletal muscle sarcoplasmic reticulum contains several SH groups which are reactive with N-ethylmaleimide (MalNEt) at pH 7.0. The location of the one which is most reactive with MalNEt (SHN, Kawakita et al. J. Biochem. 87, 609 (1980)) was identified on the amino acid sequence of the ATPase. SHN was labeled by reacting sarcoplasmic reticulum membranes with [14C] MalNEt to a labeling density of 1 mol/mol ATPase. [14C]MalNEt-labeled membranes were digested with thermolysin and 14C-labeled SHN peptides were fractionated by Sephadex LH-20 chromatography to give two major peaks of radioactivity. [14C]-MalNEt-labeled peptides were further purified to homogeneity by C18-reversed phase HPLC. Two radioactive peptides containing modified cysteine (Cys), Leu-Gly-Cys-Thr-Ser and Val-Cys-Lys-Met, were finally obtained in roughly equal amounts and in reasonable recovery. Both of these sequences were found in the amino acid sequence of Ca2+-transporting ATPase (Brandl et al. Cell 44, 597 (1986)), and Cys344 and Cys364 were identified as the targets of MalNEt-modification. Thus, 0.5 mol/mol ATPase of each Cys residue actually reacted rapidly with MalNEt under the conditions leading to SHN-modification. Modification of either one with MalNEt may negatively affect the reactivity of the other. Both of the highly reactive SH groups are located in the neighborhood of Asp351, the phosphorylation site of ATPase.