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.     

Diagnosis of a case of pulmonary carcinosarcoma by detection of rhabdomyosarcoma cells in sputum

Cytologic examination of sputum samples from an elderly patient revealed the presence of two cell populations: squamous cell carcinoma cells and rhabdomyosarcoma cells. The abnormal squamous cells showed both keratinizing and nonkeratinizing forms while some of the rhabdomyosarcoma cells showed cross striations. Sputum cytology was thus able to suggest a diagnosis of pulmonary carcinosarcoma. Histologically, the tumor was composed mainly of sarcomatous tissue showing various kinds of cells: fusiform or fibrous cells, round anaplastic cells, spindled cells with typical cross striations and myoblastic cells. A partially myxomatous degeneration was present. In addition, squamous cell carcinoma proliferated along the bronchi and formed small invasive cell nests in the sarcomatous tissue. No transition between the two components was noted. Both cellular constituents had metastasized to an interlobar lymph node.     

Benzodiazepines and their metabolites: relationship between binding affinity to the benzodiazepine receptor and pharmacological activity

Experiments were carried out to study the relationship between binding affinity to the benzodiazepine receptor and pharmacological activity, especially anti-anxiety activity, of clinically useful benzodiazepines. In the in vitro experiments, fludiazepam showed the highest affinity to the benzodiazepine receptor with 4 times more potency than that of diazepam, which paralleled the in vivo activity. Diazepam and nimetazepam also bound with high affinities as expected from their in vivo activities. On the contrary, medazepam and cloxazolam showed extremely low affinities and oxazolam showed no affinity, although they showed moderate in vivo activity. However, their metabolites were found to have both high affinity and in vivo activities. These results strongly suggest that in the case of medazepam, cloxazolam and oxazolam, their metabolites may bind to receptor sites in the brain and then elicit pharmacological action. This conclusion was supported by the fact that a good correlation between the binding affinity and the anti-anxiety activity of the tested compounds was observed.     

A new hereditary single band variant of the Gc system

Summary A new single band variant (Gc Ar) or the Gc subtypes not identical with the known Gc variants has been detected in the plasma of a healthy blood donor by isoelectric focusing. Using this technique the variant is represented by a single band which has a similar isoelectric point to the Gc 1C2 anodal band. It is well known that the single band Gc phenotypes remain unaltered after neuraminidase treatment. Nevertheless, the new single band variant (Gc Ar) is altered after neuraminidase treatment as is Gc 2A3. After neuraminidase treatment, the Gc Ar band is affected and moved to the nearby position of the Gc 2 band. Investigation of the proband's family shows that the variant occurs combined with the common alleles Gc 1F, Gc 1S and that it has an autosomal dominant inheritance.     

A novel complex mutation in the LDL receptor gene probably caused by the simultaneous occurrence of deletion and insertion in the same region

A novel complex mutation with the presence of both deletion and insertion in very close proximity in the same region was detected in exon 8 of the LDL receptor gene from two apparently unrelated Japanese families with familial hypercholesterolemia (FH). In this mutant LDL receptor gene, the nine bases from nucleotide (nt) 1115 to nt 1123 (AGGGTGGCT) were replaced by six different bases (CACTGA), and consequently the four amino acids from codon 351 to 354, Glu-Gly-Gly-Tyr, were replaced by three amino acids, Ala-Leu-Asn, in the conserved amino acid region of the growth factor repeat B of the LDL receptor. The nature of the amino acid substitution and data on the families suggest that this mutation is very likely to affect the LDL receptor function and cause FH. The generation of this complex mutation can be explained by the simultaneous occurrence of deletion and insertion through the formation of a hairpin-loop structure mediated by inverted repeat sequences. Thus this mutation supports the hypothesis that inverted repeat sequences influence the stability of a given gene and promote human gene mutations.     

Effects of fenvalerate and esfenvalerate on hepatic gap junctional intercellular communication in rats

Effects of in vivo exposure with fenvalerate, esfenvalerate andDDT on hepatic gap junctional intercellular communication (GJIC) in Sprague-Dawley (SD) rats were examined by in vivolin vitro dye-transfer assay and by immunohistochemical staining of connexin 32 (C×32, major liver gap junction protein). Fenvalerate (75 mg/kg/day), esfenvalerate (25 mg/kg/day), DDT (50 mg/kg/day) and corn oil (vehicle control, 5mllkglday) were administered orally once a day. Animals were killed at weeks 1, 2, 4 and 6 after starting the experiment. In the fenvalerate- and esfenvalerate-groups, no compound-related changes in GJIC and C×32 expression were observed. On the contrary, in the DDT-group, average sizes of the dye spread after injection of Lucifer Yellow decreased at weeks 1, 2 and 4, and the area per GJ spot shown by C×32-immunohistochemical staining decreased at weeks 4 and 6. It is concluded that neither fenvalerate nor esfenvalerate inhibits hepatic GJIC with in vivo exposure.     

The mechanism of the movement of leucocytes

In a study of the movement of human leucocytes it was clarified that characteristic contraction waves were observed on the cell surface during movement and an initial morphological change directly related to the appearance of the wave originated in the surface of the granuloplasm and not in the cell membrane. From these findings, together with physicochemical properties of the contractile protein from equine leucocytes, it was proposed that the wave observed in moving leucocytes might be conducted, in some way, by contraction and relaxation of the contractile protein in the cells. Myosin A and actin as constituents of the contractile protein were extracted separately from leucocytes in polymerized form, which resemble myosin aggregate and F-actin from muscle, respectively. The thick and thin filaments of about 150 and 80 Å in diameter were observed in glycerinated leucocytes with electron microscopy. When glycerinated leucocytes were incubated with heavy meromyosin (HMM) from rabbit skeletal myosin A, the thin filaments developed a structure resembling the ‘arrowhead structure’ of the HMM F-actin complex in vitro. The thick filaments seemed to correspond to myosin aggregates and the thin ones to filaments containing F-actin.     

Secretion of DNA synthesis factor (DSF) by A431 cells that can grow in protein-free medium

A431 cells grew in protein-free Coon's modified Ham's F12 medium at a similar rate to that in medium supplemented with calf serum and secreted a growth factor capable of stimulating DNA synthesis in BALB/c3T3 cells. This factor had strong affinity for heparin and was partially purified from the conditioned medium by heparin-Sepharose affinity chromatography and molecular sieving on Bio-Gel P-60. The apparent molecular weight of the factor was 20-30K. Its activity was inhibited by heparin at concentrations of above 0.03 microgram/ml.     

Intraspecies host specificity of a single-stranded RNA virus infecting a marine photosynthetic protist is determined at the early steps of infection

Viruses are extremely abundant in seawater and are believed to be significant pathogens to photosynthetic protists (microalgae). Recently, several novel RNA viruses were found to infect marine photosynthetic protists; one of them is HcRNAV, which infects Heterocapsa circularisquama (Dinophyceae). There are two distinct ecotypes of HcRNAV with complementary intraspecies host ranges. Nucleotide sequence comparison between them revealed remarkable differences in the coat protein coding gene resulting in a high frequency of amino acid substitutions. However, the detailed mechanism supporting this intraspecies host specificity is still unknown. In this study, virus inoculation experiments were conducted with compatible and incompatible host-virus combinations to investigate the mechanism determining intraspecies host specificity. Cells were infected by adding a virus suspension directly to a host culture or by transfecting viral RNA into host cells by particle bombardment. Virus propagation was monitored by Northern blot analysis with a negative-strand-specific RNA probe, transmission electron microscopy, and a cell lysis assay. With compatible host-virus combinations, propagation of infectious progeny occurred regardless of the inoculation method used. When incompatible combinations were used, direct addition of a virus suspension did not even result in viral RNA replication, while in host cells transfected with viral RNA, infective progeny virus particles with a host range encoded by the imported viral RNA were propagated. This indicates that the intraspecies host specificity of HcRNAV is determined by the upstream events of virus infection. This is the first report describing the reproductive steps of an RNA virus infecting a photosynthetic protist at the molecular level.