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.     

Characterisation of Trypanosoma cruzi populations by dna polymorphism of the cruzipain gene detected by single-stranded dna conformation polymorphism (SSCP) and direct sequencing

Fifty fresh isolates of Trypanosoma cruzi from Triatoma dimidiata vectors and 31 from patients with Chagas disease were analysed for DNA polymorphisms within the 432-bp core region of the cruzipain gene which encodes the active site of cathepsin L-like cystein proteinase. The cruzipain gene showed signs of polymorphism consisting of four different DNA sequences in Central and South American isolates of T. cruzi. The PCR fragments of Guatemalan isolates could be divided into three groups, Groups 1, 2 and 3, based on different patterns of single-stranded DNA conformation polymorphism. All of the strains isolated from Brazil, Chile, and Paraguay, except for the CL strain, showed a Group 4 pattern. Two to four isolates from each group were analysed by cloning and sequencing. A silent mutation occurred between Groups 1 and 2, and five nucleotides and two aa substitutions were detected between Groups 1 and 3. The DNA sequence of Group 4 contained five nucleotides and one aa substitution from Group 1. All of the DNA sequences corresponded well with the single-stranded DNA conformation polymorphism. The Group 1 isolates, the majority in the Guatemalan population (70/81, 86.4%), were isolated from both triatomines and humans, but Group 3 were isolated only from humans. Moreover, the Group 2 isolates were detected only in triatomine vectors (9/50; 18%), but never in humans (0/32, P<0.05) suggesting that this group has an independent life-cycle in sylvatic animals and is maintained by reservoir hosts other than humans.     

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.     

Studies on the formation and stability of a complex between Streptomyces proteinaceous metalloprotease inhibitor and thermolysin.

The effects of certain physicochemical parameters on the formation and stability of a complex between Streptomyces proteinaceous metalloprotease inhibitor (SMPI) and thermolysin were investigated. SMPI had its lowest Ki value at a pH of around 6.5 (similar to the pH dependence of the kcat/K(m) of thermolysin catalysis), reflecting the splitting mechanism of the SMPI inhibition of thermolysin. This Ki increased with an increase in pressure, and in (Ki-1) was almost linear with respect to pressure. The volume of the reaction (delta Vcomp), which is the volume change accompanying enzyme-inhibitor complex formation, was calculated as +8.1 +/- 0.3 mL.mol-1, which has a sign opposite to delta Vcomp for neutral peptide inhibitors and acyl-peptide substrates. The temperature dependence of Ki-1 gave the reaction enthalpy (delta Hcomp) and reaction entropy (delta Scomp) of the complex formation as 34.6 +/- 1.4 kJ.mol-1 and 298 +/- 5 J.mol-1.K-1, respectively. These positive reaction volumes and reaction entropies were related to the electrostatic interactions and ionic strength dependence of Ki which corresponded to the key ionic interaction during complex formation. Complex formation with SMPI stabilized thermolysin against pressure perturbation as observed by the changes in the Trp fluorescence of thermolysin with increasing pressure. Thermal stability, however, was affected very little by complex formation with SMPI. Phosphoramidon, Cbz-Phe-Gly-NH2 and Cbz-Phe also positively affected the pressure-tolerance of thermolysin, in the following order: Cbz-Gly-Phe-NH2 < Cbz-Phe < phosphoramidon. The third compound exhibited stabilizing effects comparable with those of SMPI, which suggests that the interaction between SMPI and thermolysin was localized to the reactive site.     

The structure of B-helical C-G-A-T-C-G-A-T-C-G and comparison with C-C-A-A-C-G-T-T-G-G. The effect of base pair reversals

The crystal structure of the DNA decamer C-G-A-T-C-G-A-T-C-G has been solved to a resolution of 1.5 A, with a final R-factor of 16.1% for 5,107 two-sigma reflections. Crystals are orthorhombic space group P2(1)2(1)2(1), with cell dimensions a = 38.93 A, b = 39.63 A, c = 33.30 A, and 10 base pairs/asymmetric unit. The final structure contains 404 DNA atoms, 142 water molecules treated as oxygen atoms, and two Mg(H2O)6(2+) complexes. Decamers stack atop one another to simulate continuous helical columns through the crystal, as with three previously solved monoclinic decamers, but the lateral contacts between columns are quite different in the orthorhombic and monoclinic cells. Narrow and wide regions of the minor groove exhibit a single spine or two ribbons of hydration, respectively, and the minor groove is widest when BII phosphate conformations are opposed diagonally across the groove. Phosphate conformation, in turn, appears to have a base sequence dependence. Twist, rise, cup, and roll are linked as has been observed in the three monoclinic decamers and can be characterized by high or low twist profiles. In all five known decamer crystal structures and eight representative dodecamers, a high twist profile is observed with G-C and G-A steps whereas all other R-R steps are low twist profiles (R = purine). A-T and A-C steps are intermediate in character whereas C-A and C-G exhibit behavior that is strongly influenced by the profiles of the preceding and following steps. When sufficient data are in hand, sequence/structure relationships for all helix parameters probably should be considered in a 4-base pair context. At this stage of limited information the problem is compounded because there are 136 unique 4-base steps x-A-B-y in a double helix as compared with only 10 2-base steps A-B.     

Polymorphism of T-cell receptor genes among laboratory and wild mice: Diverse origins of laboratory mice

Southern blots of genomic DNA from 23 strains of laboratory mice and 19 individual wild mice were examined for restriction fragment length polymorphisms in their loci encoding the T-cell receptors (Tcr): the constant regions of the α, β, and γ chains (C _α,C _β, andC _γ) and a variable region family of the β chain (V _β8). Only a few polymorphisms were observed for each locus in the laboratory mice after using three restriction enzymes,Bam HI,Eco RI, andHind III. All the laboratory mice examined fall into one of two types for theC _α,C _β andV _β8 loci and one of three types for theC _γ. These types are found in some of the wild mice studied, indicating that they were already present in the founder mice of laboratory mouse strains. In contrast, theTcr genes are highly polymorphic among wild mice. Analysis of the polymorphisms in these loci suggests that laboratory mice have inherited their genes not only fromMus musculus domesticus, but also from other subspecies, and much more than previously believed from Asian subspecies.     

Stabilization of cortical microtubules by the cell wall in cultured tobacco cells

Cortical microtubules (MTs) in protoplasts prepared from tobacco (Nicotiana tabacum L.) BY-2 cells were found to be sensitive to cold. However, as the protoplasts regenerated cell walls they became resistant to cold, indicating that the cell wall stabilizes cortical MTs against the effects of cold. Since poly-l-lysine was found to stabilize MTs in protoplasts, we examined extensin, an important polycationic component of the cell wall, and found it also to be effective in stabilizing the MTs of protoplasts. Both extensin isolated from culture filtrates of tobacco BY-2 cells and extensin isolated in a similar way from cultures of tobacco XD-6S cells rendered the cortical MTs in protoplasts resistant to cold. Extensin at 0.1 mg·ml⁻¹ was as effective as the cell wall in this respect. It is probable that extensin in the cell wall plays an important role in stabilizing cortical MTs in tobacco BY-2 cells.     

A cis-acting element and a trans-acting factor involved in the wound-induced expression of a horseradish peroxidase gene

The mechanisms that control the wound-induced expression of the prxC2 gene for horseradish peroxidase (HRP) have been investigated. Analysis of the regulatory properties of 5′-deleted promoters showed that a positive element involved in the response to wounding was located between −307 and −99 bp from the site of initiation of translation. In in vitro binding assays of tobacco nuclear proteins and DNA fragments of prxC2 promoter, the binding site was the Box 1 from −296 to −283 containing the CACGTG motif. To identify the functional role of Box 1, the prxC2 promoter that has been digested from the 5′ end to −289 with a disrupted Box 1 was fused to a reporter gene for β-glucuronidase (GUS). No induction of GUS activity was observed in transgenic tobacco plants with the prxC2(−289)/GUS construct. These data indicated that the expression of prxC2 in response to wounding required the Box 1 sequence from −296 to −283. Furthermore, a tobacco cDNA expression library was screened and a cDNA clone for a protein, designated TFHP-1, that bound specifically to the Box 1 sequence was identified. The putative TFHP-1 protein contains a basic region and leucine zipper (bZip) motif and a helix—loop—helix (HLH) motif. The mRNA for TFHP-1 was abundant in roots and stems, and it was not induced by wounding in leaves. In tobacco protoplasts, antisense TFHP-1 suppressed the expression of prxC2 (−529)/GUS.