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.     

Aggregatibacter actinomycetemcomitans induces detachment and death of human gingival epithelial cells and fibroblasts via elastase release following leukotoxin‐dependent neutrophil lysis

Aggregatibacter actinomycetemcomitans is considered to be associated with periodontitis. Leukotoxin (LtxA), which destroys leukocytes in humans, is one of this bacterium's major virulence factors. Amounts of neutrophil elastase (NE), which is normally localized in the cytoplasm of neutrophils, are reportedly increased in the saliva of patients with periodontitis. However, the mechanism by which NE is released from human neutrophils and the role of NE in periodontitis is unclear. In the present study, it was hypothesized that LtxA induces NE release from human neutrophils, which subsequently causes the breakdown of periodontal tissues. LtxA‐treatment did not induce significant cytotoxicity against human gingival epithelial cells (HGECs) or human gingival fibroblasts (HGFs). However, it did induce significant cytotoxicity against human neutrophils, leading to NE release. Furthermore, NE and the supernatant from LtxA‐treated human neutrophils induced detachment and death of HGECs and HGFs, these effects being inhibited by administration of an NE inhibitor, sivelestat. The present results suggest that LtxA mediates human neutrophil lysis and induces the subsequent release of NE, which eventually results in detachment and death of HGECs and HGFs. Thus, LtxA‐induced release of NE could cause breakdown of periodontal tissue and thereby exacerbate periodontitis.     

Immunologic characterization and molecular profile of carcinoembryonic antigen detected by monoclonal antibodies

Four distinct monoclonal antibodies, which reacted with CEA preparations but not with nonspecific cross-reacting antigen or with nonspecific cross-reacting antigen 2, were established. Except for monoclonal antibody AS001 , all of these monoclonal antibodies immunoprecipitated molecular forms of 200K and 180K daltons that are not bridged by disulfide bonds. Immunodepletion experiments and sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis revealed that these monoclonal antibodies recognized the same antigenic structure when 125I-CEA preparation was used. Monoclonal antibody AS001 is of particular interest, because this antibody reacted only with a 200K dalton molecule which is a part of the molecules recognized by the other three monoclonal antibodies. The rosette inhibition assay and the immunoprecipitation experiments suggest that each monoclonal antibody recognizes a different antigenic determinant. The antigenic determinants recognized by monoclonal antibodies YK013 and AS001 may be peptides in nature, whereas the determinants recognized by antibodies YK024 or AS005 might be carbohydrate. The radioimmunoassay with monoclonal antibody AS001 was established, and the results clearly indicate that the incidence of positivity for the sera from digestive tract cancer patients and from lung cancer patients obtained by monoclonal antibody AS001 was higher than that obtained by the polyclonal antibody. Monoclonal antibody AS001 was able to detect the corresponding antigen in the sera, which the polyclonal antibody failed to detect. This study therefore suggests that monoclonal antibodies may enhance and improve the diagnostic value in cancer patients with undetectable or lower CEA levels detected by conventional anti-CEA antibodies.     

A Bacillus subtilis dnaG mutant harbours a mutation in a gene homologous to the dnaN gene of Escherichia coli

A dnaG mutation of Bacillus subtilis, dnaG5, was found to be linked closely to recF. We have reported previously that two putative dna genes, 'dnaA' and 'dnaN', highly homologous to Escherichia coli's dnaA and dnaN, respectively, were located adjacent to recF [Ogasawara et al., EMBO J., 4 (1985) 3345-3350]. Transformation by various fragments cloned from the 'dnaA'-recF region of the wild-type cell revealed that a 532-bp AluI fragment containing 5'-portion of the 'dnaN' gene could transform the dnaG5 mutation. The nucleotide (nt) sequence of the same fragment cloned from the mutant cell shows a single nt change in the ORF of 'dnaN' which in turn causes a single amino acid alteration from Gly to Arg. The 'dnaN' gene is now proven to be a dna gene, mutations in which result in instant arrest of chromosomal replication.     

Further mutagenicity studies on pesticides in bacterial reversion assay systems

A total of 228 pesticides (88 insecticides, 60 fungicides, 62 herbicides, 12 plant-growth regulators, 3 metabolites and 3 other compounds) was tested for mutagenicity in bacterial reversion-assay systems with 5 strains (TA100, TA98, TA1535, TA1537 and TA1538) of Salmonella typhimurium and a strain (WP2 hcr) of Escherichia coli. 50 pesticides (25 insecticides, 20 fungicides, 3 herbicides, 1 plant-growth regulator and 1 other compound) were found to be mutagenic. 5 of them required metabolic activation (S9 mix) for their activities. Among various chemical groups, organic phosphates, halogenated alkanes and dithiocarbamates showed higher ratios of mutagens. Although 22 of the pesticides tested have been reported to be carcinogenic, 7 of them, i.e., captain, DBCP, EDB, EDC, ETU, HEH and nitrofen, were detected as mutagens in the present assay. Most of the other 15 non-mutagenic carcinogens were organochlorine pesticides such as alpha-BHC, chlorobenzilate, p,p'-DDT, dieldrin and quintozene.     

Antimutagenic effects of cinnamaldehyde on chemical mutagenesis in Escherichia coli

Antimutagenic effects of cinnamaldehyde on mutagenesis by chemical agents were investigated in Escherichia coli WP2 uvrA- trpE-. Cinnamaldehyde, when added to agar medium, greatly reduced the number of Trp+ revertants induced by 4-nitroquinoline 1-oxide (4-NQO) without any decrease of cell viability. This antimutagenic effect could not be explained by inactivation of 4-NQO caused by direct interaction with cinnamaldehyde. Mutagenesis by furylfuramide (AF-2) was also suppressed significantly. Mutations induced by methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS) were slightly inhibited. However, cinnamaldehyde was not at all effective on the mutagenesis of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Two derivatives of cinnamaldehyde, cinnamyl alcohol and trans-cinnamic acid, did not have as strong antimutagenic effects on 4-NQO mutagenesis as cinnamaldehyde had. Because cinnamaldehyde showed marked antimutagenic effects against mutations induced by UV-mimic mutagens but not those induced by MNNG or EMS, it seems that cinnamaldehyde might act by interfering with an inducible error-prone DNA repair pathway.     

Ontogeny of lymphocytes expressing J chain in chickens

The ontogeny of chicken lymphocytes expressing J chain (LEJ) was investigated in the embryonic bursa of Fabricius, the spleen, and the thymus. Simultaneous appearance of LEJ was detected in the bursa and spleen on Day 14 of incubation. These cells were detected later in the thymus. The LEJ were found to increase rapidly in the spleen from the 19th to 20th incubation day. In adult chickens, the highest percentage of LEJ was also found in the spleen. These cells were seen in the thymus at a lower frequency. Intermediate numbers were found in bursal and peripheral blood lymphocytes. The frequencies of the LEJ were similar to those of lymphocytes positive for cytoplasmic immunoglobulins (Ig) IgA and IgM, but were not related to the number of lymphocytes expressing surface Ig. It is possible to consider that the suitable site for LEJ is the spleen, on the basis of the rapid increase in the number of LEJ just before hatching and from the fact that the highest value is found in adult chickens. Furthermore, LEJ may participate in secretion of IgA or IgM but not be associated with the expression of surface Ig.