Antibody response after varicella vaccination in children treated with budesonide inhalation suspension or non-steroidal conventional asthma therapy.

We evaluated if budesonide inhalation suspension (BIS) reduces the immunogenicity of the varicella vaccine in paediatric patients with asthma. This open-label, parallel-group, cohort study included varicella-naïve (disease and vaccine) children aged 12 months to 8 years with asthma requiring therapy. Patients who received > or = 4 weeks of asthma treatment with BIS 0.25-1 mg daily or non-steroidal conventional asthma therapy (NSCAT) daily or as needed and met eligibility requirements received the varicella vaccine (Varivax) and continued the same asthma treatment for > or = 8 weeks postvaccination. Varicella-zoster virus (VZV) antibody levels were assessed before and 6 weeks after vaccination using a glycoprotein enzyme-linked immunosorbent assay (gpELISA). Adverse events (AEs) were assessed throughout the study. Antibody levels were analysed in 243 of 274 patients who were vaccinated and received treatment. After immunisation, the percentage of patients in each group achieving a 'protective' level of VZV antibody (> or = 5 gpELISA units/ml) was similar: 85% (129/151) in the BIS group and 90% (83/92) in the NSCAT group (relative risk = 0.95; 95% confidence interval 0.86-1.04). Eight patients in each group reported AEs related to varicella vaccination (primarily pyrexia, agitation and injection-site reactions). There were no cases of severe varicella in either group; one case of mild varicella-like rash was reported in a 12-month-old child in the NSCAT group 11 days after vaccination. VZV antibody responses and tolerability to the live varicella vaccine in paediatric asthma patients treated with BIS vs. NSCAT were comparable, demonstrating that young children with asthma receiving nebulised BIS can be immunised effectively with Varivax.

Myocardial mRNA content and stability, and enzyme activities of Ca-cycling and aerobic metabolism in canine dilated cardiomyopathies.

Idiopathic dilated cardiomyopathy is associated with derangement of myocardial sarcoplasmic Ca-homeostasis and energy production. The molecular mechanism for these changes is unknown. Accordingly, we used genetic and experimentally-induced models of canine dilated cardiomyopathy and tested the hypothesis that these metabolic changes resulted from altered gene expression, as indicated by mRNA content. We studied dilated cardiomyopathy occurring naturally (n = 9) in Doberman pinschers, and in dogs subjected to rapid ventricular pacing (n = 5), in comparison with normal dogs (n = 9). We determined content and integrity of mRNA's using Northern and slot blotting, and measured activities of their translated product for the Ca-release channel and Ca-ATPase of sarcoplasmic reticulum, lactate dehydrogenase of glycolysis, citrate synthase of the tricarboxylic acid cycle, and for myoglobin, ATP-synthetase and the adenine nucleotide transporter, which are integral in oxidative phosphorylation. We found that, whereas both mRNA content and enzyme activity for markers of Ca-cycling, glycolysis, and oxidative phosphorylation were downregulated (20-80%) in dilated cardiomyopathy, they were upregulated (10-15%) for tricarboxylic acid cycling and for ribosomal RNA. RNA from cardiomyopathic tissue was up to 50% more degraded than for normal hearts in association with a 150% increase in ribonuclease activity. Downregulation of the Ca-cycle was asymmetric, with the Ca-channel being 65% more affected than the Ca-ATPase. This work supports the general paradigm that transcriptional and translational responses to pathophysiology are major determinants of the metabolic response seen in cardiac failure.

Conformation states of Xenopus transcription factor IIIA.

The conformation of Xenopus transcription factor IIIA (TFIIIA) free in solution, bound to 5S RNA in the 7S particle, depleted of zinc, or bound to plasmid DNA was analyzed by (1) trypsin digestion and electrophoretic analysis of proteolytic fragments or (2) measurement of the fluorescence of TFIIIA mildly derivatized with N-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonic acid (IAEDANS). TFIIIA free or complexed with 5S RNA has a similar conformation as judged (a) by trypsin-dependent generation of similar metastable 20-kDa domains (corresponding to the N-terminal half of the protein) or (b) by the negligible change in AEDANS-TFIIIA fluorescence when free or bound to 5S RNA. When TFIIIA binds plasmid DNA, its N-terminal half becomes hypersensitive to trypsin digestion, indicating a structural change in this region of the protein upon interaction with DNA. Quenching of AEDANS-TFIIIA fluorescence is observed upon interaction of the protein with plasmid DNA, a result also indicative of a conformational change upon protein-DNA interaction. Removal of zinc from TFIIIA by EDTA chelation results in (a) increased proteolysis of this 20-kDa domain, indicating a structural change in the N-terminal half of the protein upon zinc removal, and (b) large enhancement of AEDANS-TFIIIA fluorescence. EDTA chelation of TFIIIA bound to 5S RNA in the 7S particle, a procedure which does not deplete all zinc from the protein, neither increases the trypsin sensitivity of the 20-kDa domain nor alters appreciably the fluorescence of AEDANS-TFIIIA. These results indicate that zinc is involved in maintaining the native conformation of at least the N-terminal half of the protein.

Species variation in transcription factor IIIA.

Species variation in transcription factor IIIA (TFIIIA) was examined by comparing the abilities of TFIIIAs isolated from different Xenopus and Rana species to 1) bind rabbit anti-Xenopus laevis TFIIIA IgG, 2) specifically interact with the Xenopus borealis somatic 5S RNA gene, and 3) promote transcription of the Xenopus borealis 5S RNA gene in vitro. In immunoblot assays, Rana catesbeiana or Rana pipiens TFIIIA did not react readily with rabbit anti-Xenopus laevis TFIIIA IgG (assayed with anti-rabbit F(ab')2 fragment conjugated with alkaline phosphatase) whereas Xenopus borealis TFIIIA exhibited similar reactivity with this IgG as Xenopus laevis TFIIIA. When compared to Xenopus TFIIIAs, Rana TFIIIAs exhibited similar interactions with the 3' portion of the intragenic control region of the Xenopus 5S RNA gene (to residue +78 on the coding strand and up to and including +74 on the non-coding strand, nucleotides protected from DNase I digestion by the N-terminal half of Xenopus TFIIIA) and incomplete interactions with the remaining 5' portion of the control region (nucleotides protected from DNase I digestion by the C-terminal half of Xenopus TFIIIA). In a Xenopus laevis unfertilized egg extract, Rana catesbeiana and Rana pipiens TFIIIAs promoted transcription of the Xenopus borealis somatic 5S RNA gene less efficiently than Xenopus laevis and Xenopus borealis TFIIIAs.

Deletion of the N-terminal region of Xenopus transcription factor IIIA inhibits specific binding to the 5 S RNA gene.

Xenopus transcription factor IIIA (TFIIIA) is expressed in Escherichia coli by utilizing one plasmid with a T7 RNA polymerase gene and another plasmid with TFIIIA cDNA cloned downstream of a T7 promoter. Wild-type TFIIIA and a TFIIIA deletion mutant, isolated from E. coli cell extracts, are identified by antiserum against native TFIIIA purified from Xenopus immature oocytes. DNase I protection experiments indicate that wild-type TFIIIA, synthesized from a full-length TFIIIA cDNA, binds specifically to the coding and noncoding strands of the 5 S RNA gene. The TFIIIA deletion mutant, expressed from TFIIIA cDNA lacking the coding sequence for the N-terminal 29 amino acids, fails to bind specifically to the 5 S RNA gene as judged by its inability to protect to any degree the coding or noncoding strands of the gene from DNase I digestion. Both wild-type TFIIIA and the N-terminal deletion mutant promote DNA renaturation.