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.     

Disappearance of terminal deoxynucleotidyltransferase in human malignant T-lymphoblasts treated with a human alpha interferon preparation

Human T-lymphoblastoid cell lines RPMI 8402, MOLT-3, and CCRF-CEM were treated with interferon (IFN) to determine if the treatment would result in the disappearance of cellular terminaldeoxynucleotidyltransferase (TdT), a possible differentiation marker for T-lymphocytes. Incubation of RPMI 8402 cells in the presence of IFN preparation caused a decrease in the number of TdT-positive cells and in TdT activity of the cell extract. The inhibition of cell multiplication was dose dependent. The anticellular effect of IFN preparation was cytostatic, not cytocidal. The IFN preparation modified neither the TdT content nor proliferation of MOLT-3 and CCRF-CEM cell lines. The effects of IFN preparation thus varied with the cell line.     

Characterization of two types of histone H2B genes from macronuclei of Tetrahymena thermophila.

Two histone H2B gene clones were isolated from macronuclei of Tetrahymena thermophila. Nucleotide sequences of the two clones were highly homologous within the coding region but not in the noncoding region. Comparison of the deduced amino acid sequences between the two clones showed three differences in a total of 121 amino acids. Each of the two clones contained a TAA triplet within the coding region, which appeared to code for a glutamine residue. To demonstrate the existence of histone mRNA containing UAA triplet, nuclease P1 protection mapping using total cellular RNA and nucleotide sequencing of primer extension products were carried out. The results clearly indicated that two cloned histone H2B genes were transcribed, giving rise to the major histone H2B mRNAs with a UAA triplet sequence in frame. The tentative 5'- and 3'-ends of histone H2B mRNAs were determined.     

Fatty chain composition of phospholipids in sea urchin spermatozoa

1. An analysis was made of lipids extracted from the spermatozoa of the sea urchins, Hemicentrotus pulcherrimus and Anthocidaris crassispina. 2. Nearly all the lipids from both species consisted of phospholipids (about 80%) and cholesterol (about 14%). Triglyceride and cholesterol ester were present in trace amounts. 3. The fatty acid composition of each phospholipid was determined by gas-liquid chromatography. In both species, the fatty acid consisting of phosphatidylethanolamine was of the unsaturated type for the most part, while cardiolipin was comprised to a considerable degree of saturated fatty acids. In phosphatidylcholine and phosphatidylserine from H. pulcherrimus sperm, unsaturated fatty acid content was somewhat higher than that in phospholipids from A. crassispina sperm.     

Identification of (Na,K)ATPase inhibitor in brine shrimp,Artemia salina,as long-chain fatty acids

Summary An inhibitory activity to (Na,K)ATPase was found in cell extracts of the brine shrimp, Artemia salina, irrespective of its developmental stages. Organic solvent extraction together with gas chromatographic analysis reveals that the inhibitory activity is due to long-chain, non-esterified fatty acids and their derivatives. Unsaturated fatty acids, especially with cis-configuration, are more effective in inhibition than saturated ones.Abbreviations ATPase adenosine triphosphatase - EDTA ethylenediamine-tetraacetate - TLC thin-layer chromatography     

Energy metabolism of sea urchin spermatozoa, with phosphatidylcholine as the preferred substrate

Phosphatidylcholine content in the spermatozoa of the sea urchin, Hemicentrotus pulcherrimus, decreased rapidly during incubation with sea water. Sea urchin sperm contained approx. 85% phospholipid in total lipid. Phosphatidylcholine (PC) was the principal lipid. Other phospholipids, however, remained at constant levels during incubation. Although the free fatty acid content gradually increased following dilution of dry sperm in sea water, the amounts of triacylglycerol and cholesterol ester were extremely low. Analysis by gas-liquid chromatography indicated most of fatty acid moieties in PC to be polyenoic. PC composed in part of unsaturated fatty acids was consumed to a greater extent during incubation than that consisting of saturated fatty acids. Furthermore, 1-palmitoyl-2-[1-14C]linoleoylphosphatidylcholine was transformed to 14C-labelled free fatty acid in a subcellular system. Thus, possibly, phospholipase A2 is present in sea urchin sperm. Also, [1-14C]oleic acid was immediately oxidized to 14CO2 by sperm. It is thus concluded that sea urchin sperm use phosphatidylcholine as a substrate for energy metabolism.     

Characterization of human genomic DNA sequences homologous to the interleukin 2 cDNA

Southern blot analysis of human placental DNA under low stringency hybridization conditions revealed several DNA fragments hybridizable to the human interleukin 2 (IL-2) cDNA. Four phage clones carrying these IL-2 cDNA-like sequences were isolated and their structures analyzed. A DNA fragment derived from one of the clones gave the strongest hybridization signal. Sequence analysis of this fragment revealed the presence of a cluster of three DNA segments, i.e. 20 base pairs (bp), 57 bp and 18 bp in length and having about 85%, 80% and 83% homology to three different parts of the coding region of human IL-2 cDNA, respectively.