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.     

Immune deficiency in the X-linked lymphoproliferative syndrome. I. Epstein-Barr virus-specific defects

Eleven males with XLP were evaluated for EBV-specific antibodies during periods of 2 to 7 yr. Variable responses to EBV-specific antigens were found. All 11 patients had subnormal anti-EBNA titers, which probably reflected a T cell deficiency. The patients showed four different patterns in their anti-VCA response: 1) two boys who had experienced malignant lymphoma mounted no antibodies at all; 2) two patients showed intermittent anti-VCA titers; 3) four males had persistently elevated anti-VCA titers; and 4) three patients showed normal anti-VCA titers. ADCC against EBV-infected cells was abnormally low in six patients and was elevated in two patients given gamma-globulin. ADCC titers did not correlate with anti-VCA titers. However, most patients with XLP failed to effect regression of autologous EBV-infected lymphoblastoid cell lines, indicating a deficiency in long-lived T cell-mediated immunity to EBV.     

Changes in IL-6, IL-8, C-reactive protein and pancreatic secretory trypsin inhibitor after transcatheter arterial chemo-embolization therapy for hepato-cellular carcinoma.

In an attempt to investigate the interaction between the changes of cytokines and acute phase reactants after transcatheter arterial chemoembolization therapy (TACE), the levels of interleukin 6 (IL-6), interleukin 8 (IL-8), C-reactive protein (CRP) and pancreatic secretory trypsin inhibitor (PSTI) in the blood of patients with unresectable hepatocellular carcinoma (HCC) were measured. Before the therapy, serum IL-6 and plasma IL-8 levels were detectable in 77.8% and 28.5%, respectively, of patients with HCC. Levels of serum IL-6 and plasma IL-8 increased after TACE and reached a peak on day 3 in all patients (18/18) and in 87.5% of patients (12/14), respectively. Both blood levels of IL-6 and IL-8 reached a peak earlier than those of CRP and PSTI did after the therapy. When the maximal values of IL-6 were compared with those of CRP and PSTI, there were significant positive correlations (r = 0.63, P < 0.01 and r = 0.81, P < 0.01, respectively). Similarly, comparisons of the maximal values of IL-8 with those of CRP and PSTI gave a significant correlation (r = 0.68, P < 0.01 and r = 0.67, P < 0.05, respectively). However, no significant correlation was found between the elevation of IL-6 and IL-8.     

Application of avidin-ferritin and peroxidase as contrasting electron-dense markers for simultaneous electron microscopic immunocytochemical labelling of glutamic acid decarboxylase and tyrosine hydroxylase in the rat arcuate nucleus

Summary A pre-embedding immunostaining procedure was developed using ferritin and peroxidase to enable simultaneous electron microscopic localization of two antigens in the same tissue section. This method was used to study the anatomic relationship between glutamic acid decarboxylase (GAD) immunoreactive axons and tyrosine hydroxylase (TH) — containing neurons of the rat arcuate nucleus. The findings provide ultrastructural evidence that GAD-immunoreactive terminals establish symmetric (Gray II) synapses on TH-reactive neurons.     

Immunolocalization of β-1-4-galactan and its relationship with lignin distribution in developing compression wood of Cryptomeria japonica

Compression wood (CW) contains higher quantities of β-1-4-galactan than does normal wood (NW). However, the physiological roles and ultrastructural distribution of β-1-4-galactan during CW formation are still not well understood. The present work investigated deposition of β-1-4-galactan in differentiating tracheids of Cryptomeria japonica during CW formation using an immunological probe (LM5) combined with immunomicroscopy. Our immunolabeling studies clearly showed that differences in the distribution of β-1-4-galactan between NW (and opposite wood, OW) and CW are initiated during the formation of the S₁ layer. At this stage, CW was strongly labeled in the S₁ layer, whereas no label was observed in the S₁ layer of NW and OW. Immunogold labeling showed that β-1-4-galactan in the S₁ layer of CW tracheids significantly decreased during the formation of the S₂ layer. Most β-1-4-galactan labeling was present in the outer S₂ region in mature CW tracheids, and was absent in the inner S₂ layer that contained helical cavities in the cell wall. In addition, delignified CW tracheids showed significantly more labeling of β-1-4-galactan in the secondary cell wall, suggesting that lignin is likely to mask β-1-4-galactan epitopes. The study clearly showed that β-1-4-galactan in CW was mainly deposited in the outer portion of the secondary cell wall, indicating that its distribution may be spatially consistent with lignin distribution in CW tracheids of Cryptomeria japonica.     

Precise identification of gene products in hearts after in vivo gene transfection, using Sendai virus-coated proteoliposomes.

Both efficient gene transfer and the exact identification of gene product are required for gene therapy. Gene transfection of green fluorescence protein (GFP) might be useful for the reporter. After in vivo cotransfection of GFP and beta-galactosidase (beta-Gal) genes in Sendai virus-coated proteoliposomes to rat hearts, we compared the sensitivity and specificity of three methods: GFP detection, histochemical staining (HC) of beta-Gal activity, and immunostaining (IS) of the beta-Gal protein. Fluorescence microscopy and double staining of HC and IS revealed that both GFP and IS were equally sensitive and fourfold superior to HC at the peak of gene expression. However, different from skeletal muscle, the GFP of transfected cardiomyocytes showed two demerits: the fluorescence quenching due to the intense staining of beta-Gal activity, and nonspecific autofluorescence from myocardium. Thus, specific IS would be so far the most reliable to identify the gene product in heart.     

Prophages integrating into prophages: A mechanism to accumulate type III secretion effector genes and duplicate Shiga toxin-encoding prophages in Escherichia coli

Bacteriophages (or phages) play major roles in the evolution of bacterial pathogens via horizontal gene transfer. Multiple phages are often integrated in a host chromosome as prophages, not only carrying various novel virulence-related genetic determinants into host bacteria but also providing various possibilities for prophage-prophage interactions in bacterial cells. In particular, Escherichia coli strains such as Shiga toxin (Stx)-producing E. coli (STEC) and enteropathogenic E. coli (EPEC) strains have acquired more than 10 prophages (up to 21 prophages), many of which encode type III secretion system (T3SS) effector gene clusters. In these strains, some prophages are present at a single locus in tandem, which is usually interpreted as the integration of phages that use the same attachment (att) sequence. Here, we present phages integrating into T3SS effector gene cluster-associated loci in prophages, which are widely distributed in STEC and EPEC. Some of the phages integrated into prophages are Stx-encoding phages (Stx phages) and have induced the duplication of Stx phages in a single cell. The identified attB sequences in prophage genomes are apparently derived from host chromosomes. In addition, two or three different attB sequences are present in some prophages, which results in the generation of prophage clusters in various complex configurations. These phages integrating into prophages represent a medically and biologically important type of inter-phage interaction that promotes the accumulation of T3SS effector genes in STEC and EPEC, the duplication of Stx phages in STEC, and the conversion of EPEC to STEC and that may be distributed in other types of E. coli strains as well as other prophage-rich bacterial species.     

Yeast plasmids resembling 2 micron DNA: regional similarities and diversities at the molecular level.

The nucleotide sequence of two Zygosaccharomyces plasmids, pSB2 (5,415 base pairs), isolated from Zygosaccharomyces bailii, and pSM1 (5,416 base pairs), isolated from Zygosaccharomyces fermentati Naganishi, was determined. The predicted amino acid sequences of open reading frames among six yeast plasmids that resemble 2 microns DNA indicated regional sequence similarities among FLP proteins. Greater similarities were seen among Zygosaccharomyces plasmids (pSB2, pSB3, pSR1, and pSM1) than other combinations. A putative recognition site for the FLP enzyme of a Zygosaccharomyces plasmid also showed some conservation, especially in the 4 nucleotides flanking the central spacer region. From comparative studies of the sequences of putative genes of each plasmid, we propose an apparent phylogenetic relationship among yeast plasmids resembling 2 micron DNA. Among the Zygosaccharomyces plasmids, pSB2 and pSR1 are most closely related, since not only were the FLP enzymes of these two plasmids most closely related, but also the amino acid sequence of the putative P gene of pSR1 showed clear homology with that of open reading frame B of pSB2.     

In vitro splicing of a chicken delta-crystallin pre-mRNA in a mammalian nuclear extract

An in vitro splicing system was constructed using portions of chicken delta-crystallin pre-mRNA synthesized in vitro and a HeLa nuclear extract. Analysis of the reaction products revealed that about 25% of the pre-mRNA was precisely spliced at 30 degrees C in 2 h under the standard conditions. The other major products of the reaction detected were a 5'-exon fragment and three RNA species showing unusual electrophoretic mobilities on polyacrylamide gels. Structural analyses showed that these three RNAs contain a branch (lariat) structure as seen in the in vitro splicing reactions of human beta-globin, adenovirus, and yeast pre-mRNAs. In addition, methylation at the N-7 position of the blocking guanosine of the 5'-terminal cap structure of pre-mRNA has been suggested to play an important role in the splicing reaction.