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.     

RNA-Induced Interference with Animal Virus Infection

Some RNAs, including both single- and double-stranded RNAs, when incubated with chick embryo cell culture induce cellular resistance against viruses. Evidence was now obtained indicating that the induction of cellular resistance by RNA depends on the cellular metabolic activity, especially on the synthesis of cellular RNA and protein. Thus, inhibitors of RNA and protein synthesis, actinomycin D and cycloheximide, were found to inhibit the development of an antiviral state when added before, or during the relatively early period of, incubation of the cells with RNA. In the course of induction of cellular resistance, three stages may be distinguished, the priming stage, the developing stage, and the established resistant stage.     

The critical period in which splenectomy causes functional disorder of the ovary in adult rats

On account of recent reports suggesting that ovulation and luteinization involve immunological reactions, we examined the effect of splenectomy in rats on the recurrence of an estrous cycle. The spleen was removed from adult female rats at various times during an estrous cycle. A delay in ovulation was specifically induced in the rats splenectomized on the metestrous morning between 0700 and 0900 h. This delay in ovulation was reversed by an injection of splenocytes obtained either from estrous or metestrous donors, but not from diestrous or proestrous donors. The isolated splenic macrophage preparation mimicked the effect of splenocytes. Measurement of progestin levels throughout the estrous cycle suggested that the delay in ovulation was caused primarily by a delay in luteolysis; progesterone levels in ovulation delayed rats were higher and 20 alpha-dihydroprogesterone levels were lower than those of intact rats on the diestrous day. These results suggest that the macrophages in the spleen under the influence of endocrine milieu probably play a role in the recurrence of an estrous cycle by controlling luteolysis. The specific time of splenectomy to cause delay in ovulation will afford a new approach in analyzing the function of immunocytes in the ovary.     

Immunocytochemical applications in neuroanatomy

Summary In the present paper we review immunocytochemical methods for anterograde tracing with the lectin Phaseolus vulgaris-leucoagglutinin (PHA-L), combined PHA-L tracing — neurotransmitter immunocytochemistry, and the immunocytochemical localization of receptor proteins. These methods will be mainly illustrated by examples from tracing- and neurotransmitter studies on the cholinergic basal forebrain system. The morphology of PHA-L labeled neurons strongly resembles that of Golgi impregnated neurons. The complete axonal trajectories and patterns of presynaptic endings of PHA-L labeled neurons are visualized, both for light- and electron microscopic application.PHA-L-tracing can very well be combined with second immunocytochemical labeling procedures. In this way, traced pathways can be studied in their relation to chemically identified fiber systems or target neurons. Application of immunocytochemistry for the localization of the muscarinic acetylcholine receptor, albeit in its early stages, holds great promise for the near future.     

RFP is a DNA binding protein associated with the nuclear matrix.

We reported that the RFP gene encodes a protein with putative zinc finger domains and was involved in the activation of the ret proto-oncogene. To further characterize the RFP protein, we developed a polyclonal antibody against the product synthesized from a fragment of the RFP cDNA expressed in Escherichia coli. Western blot analysis showed that RFP was identified as a 58 kDa protein in cell lysates from four human and rodent cell lines and from mouse testis. In addition, a unique 68 kDa protein was detected in the testis. Using AH7974 (rat ascites hepatoma) and Raji (human Burkitt lymphoma) cells, we demonstrated strong association of RFP with the nuclear matrix. Furthermore, RFP solubilized from the nuclear matrix had DNA-binding activity although it appears to bind more preferentially to double-stranded DNA than to single-stranded DNA. These results thus suggest that RFP may play a role in molecular processes which occur in the nuclear matrix.     

Efficient in vitro translocation into Escherichia coli membrane vesicles of a protein carrying an uncleavable signal peptide. Characterization of the translocation process

The translocation into Escherichia coli cytoplasmic membrane vesicles of a protein containing an uncleavable signal peptide was studied. The signal peptide cleavage site of the ompF-lpp chimeric protein, a model secretory protein, was changed from Ala-Ala to Phe-Pro through oligonucleotide-directed site-specific mutagenesis of the ompF-lpp gene on a plasmid. The mutant protein was no longer processed by the signal peptidase. When proteinase K treatment was adopted as a probe for protein translocation into inverted membrane vesicles, the mutant protein exhibited rapid and almost complete translocation, most likely due to the lack of premature cleavage of the signal peptide before the translocation. This result also indicates that cleavage of the signal peptide is not required for translocation of the mature domain of the protein. The establishment of an efficient system made it possible to perform precise and quantitative analysis of the translocation process. The translocation was time-dependent, vesicle-dependent, and required ATP and NADH. Translocation into membrane vesicles was also observed with the uncleavable precursor protein purified by means of immunoaffinity chromatography, although the efficiency was appreciably low. The translocation required only ATP and NADH. Addition of the cytosolic fraction did not enhance the translocation.