Hashimoto's disease is a frequent comorbidity and an exacerbating factor of chronic spontaneous urticaria

BACKGROUND: The precise pathogenesis of chronic spontaneous urticaria (CSU) remains unknown. However, an important association between CSU and autoimmune disorders such as Hashimoto's disease (HD) has been reported. We investigated the frequency of HD as a comorbidity of CSU and the prevalence rate of autoreactivity among CSU patients with HD. PATIENTS AND METHODS: The presence of thyroid autoantibodies and the levels of thyroid hormones were examined in 40 CSU patients who showed urticaria symptoms for >4 weeks. Patients who were diagnosed with HD, including subclinical ones, and were in need of treatment received thyroid therapy, and the changes in their urticarial symptoms were observed. An autologous serum skin test (ASST) was also performed to examine the relation of CSU with autoreactivity. RESULTS: Eleven of the 40 CSU patients were diagnosed with HD, and 4 of the 5 patients who received and completed thyroid therapy showed considerable remission of urticarial symptoms during and after treatment. In addition, the rate of positive ASST results tended to be higher in CSU patients with HD (5 of 7) than in those without HD (2 of 6). CONCLUSIONS: The comorbidity rate of HD in CSU patients was high, and such patients tended to have a positive ASST. Thyroid therapy in CSU patients with HD can lead to a considerable remission of urticarial symptoms, which may suggest that HD is possibly involved in the aetiology of CSU, or is at least a potential exacerbating factor for CSU

No disponible

Modeling of the rf discharge initiation in a negative ion source.

The maintenance free rf ion source is expected to be one of the most promising candidates for the negative ion sources of plasma heating for future fusion reactors. As an alternative to the arc-discharge sources, the rf negative ion sources have been developed for H(-) production. In order to make clear the condition for the discharge initiation of the rf source, we are developing a numerical model using the finite difference time domain Monte Carlo method to analyze the electron energy distribution function in rf field. The numerical result shows that the discharge is not successfully initiated due to the wall loss unless the wall potential is considered. More self-consistent model including ion dynamics to evaluate the wall potential and the electron loss at the wall will be needed in the future.

Autonomous role of 3'-terminal CCCA in directing transcription of RNAs by Qbeta replicase.

We have studied transcription in vitro by Qbeta replicase to deduce the minimal features needed for efficient end-to-end copying of an RNA template. Our studies have used templates ca. 30 nucleotides long that are expected to be free of secondary structure, permitting unambiguous analysis of the role of template sequence in directing transcription. A 3'-terminal CCCA (3'-CCCA) directs transcriptional initiation to opposite the underlined C; the amount of transcription is comparable between RNAs possessing upstream (CCA)(n) tracts, A-rich sequences, or a highly folded domain and is also comparable in single-round transcription assays to transcription of two amplifiable RNAs. Predominant initiation occurs within the 3'-CCCA initiation box when a wide variety of sequences is present immediately upstream, but CCA or a closely similar sequence in that position results in significant internal initiation. Removal of the 3'-A from the 3'-CCCA results in 5- to 10-fold-lower transcription, emphasizing the importance of the nontemplated addition of 3'-A by Qbeta replicase during termination. In considering whether 3'-CCCA could provide sufficient specificity for viral transcription, and consequently amplification, in vivo, we note that tRNA(His) is the only stable Escherichia coli RNA with 3'-CCCA. In vitro-generated transcripts corresponding to tRNA(His) served as poor templates for Qbeta replicase; this was shown to be due to the inaccessibility of the partially base-paired CCCA. These studies demonstrate that 3'-CCCA plays a major role in the control of transcription by Qbeta replicase and that the abundant RNAs present in the host cell should not be efficient templates.

Internal and 3' RNA initiation by Qbeta replicase directed by CCA boxes.

RNA initiation by Qbeta replicase directed by the short-sequence CCA at the 3'-end of all RNAs amplified by this enzyme has been studied. Most CCA repeats in an RNA consisting of 12 CCAs serve as independent sites of de novo RNA initiation, with initiation occurring opposite the 3'-C residue of each CCA. Qbeta replicase is thus capable of internal initiation remote from the 3'-end, although predominant initiation occurs close to the 3'-end. The precise 3'-terminal sequence in (CCA)(n)-containing RNAs influences the number and position of active initiation sites near the 3'-terminus. C residues are required at the initiation site, whereas the position of purines (especially A residues) influences the selection of initiation sites. The template activity of (CCA)(n) RNAs is positively correlated with the number of CCA repeats. Three CCA repeats added to the 3'-end of a nontemplate 83-nt RNA are sufficient to activate transcription by Qbeta replicase. These experiments show that CCA boxes can act as strong initiation sites in the absence of specific cis-acting signals derived from Qbeta RNA, although the efficiency of initiation is modulated by surrounding sequence.

CCA initiation boxes without unique promoter elements support in vitro transcription by three viral RNA-dependent RNA polymerases.

It has previously been observed that the only specific requirement for transcriptional initiation on viral RNA in vitro by the RNA-dependent RNA polymerase (RdRp) of turnip yellow mosaic virus is the CCA at the 3' end of the genome. We now compare the abilities of this RdRp, turnip crinkle virus RdRp, and Qbeta replicase, an enzyme capable of supporting the complete viral replication cycle in vitro, to transcribe RNA templates containing multiple CCA boxes but lacking specific viral sequences. Each enzyme is able to initiate transcription from several CCA boxes within these RNAs, and no special reaction conditions are required for these activities. The transcriptional yields produced from templates comprised of multiple CCA or CCCA repeats relative to templates derived from native viral RNA sequences vary between 2:1 and 0.1:1 for the different RdRps. Control of initiation by such redundant sequences presents a challenge to the specificity of viral transcription and replication. We identify 3'-preferential initiation and sensitivity to structural presentation as two specificity mechanisms that can limit initiation among potential CCA initiation sites. These two specificity mechanisms are used to different degrees by the three RdRps. The finding that three viral RdRps representing two of the three supergroups within the positive-strand RNA viral RdRp phylogeny support substantial transcription in the absence of unique promoters suggests that this phenomenon may be common among positive-strand viruses. A framework is presented arguing that replication of viral RNA in the absence of unique promoter elements is feasible.

A novel cell-free system for peptide synthesis driven by pyridine.

Previously we demonstrated that ribosomes can synthesize polypeptides in the presence of high concentrations (40-60%) of pyridine without any protein factors. Here we analyze additional ribosomal parameters in 60% pyridine using Escherichia coli ribosomes. Ribosomal subunits once exposed to pyridine failed to re-associate to 70S ribosomes in aqueous buffer systems even in the presence of 20 mM Mg2+, whereas they formed 70S complexes in the presence of 60% pyridine. Two-dimensional gel electrophoresis of ribosomal proteins revealed that some proteins located at the protuberances of the large subunit, e. g. L7/L12 and L11 forming the elongation factor-binding domain, were released in the pyridine system. The aminoglycoside neomycin, a strong inhibitor of the ribosomal (factor-independent) translocation reaction, completely blocked poly(Phe) synthesis and translocation activities in the pyridine system, whereas these activities were not affected at all by gypsophilin, a ribotoxin that inhibits factor-dependent translocation. Another inhibitor of the ribosomal translocation, thiostrepton, had no effect concerning the two activities, which is consistent with the fact that this antibiotic requires L11 for its binding to the ribosome. These results suggest that the ribosomes can perform a translocation reaction in the pyridine system, but in a factor-independent (spontaneous) manner.

Gypsophilin, a new type 1 ribosome-inactivating protein from Gypsophila elegans: purification, enzymatic characterization, and subcellular localization.

Gypsophila elegans contains a new type 1 ribosome-inactivating protein, which we named gypsophilin. The protein was purified to apparent homogeneity by (NH4)2SO4 fractionation, ion-exchange chromatography, and adsorption chromatography. The protein was found to have a molecular mass of 28.0 kDa and a pI of about 10.1. It does not contain glycosidic linkages. The sequence of the N-terminal 22 amino acids of the protein shows a close relationship to other RIPs. The enzyme strongly inhibits protein synthesis in a rabbit reticulocyte lysate and depurinates 28S rRNA in rat liver ribosomes in a manner identical to that of ricin A-chain and other RIPs. Using a direct method for the measurement of the RNA N-glycosidase activity, the substrate specificity of gypsophilin was identified. EC50 of the protein for ribosomes of rat liver, wheat germ, and E. coli was 39.8 pM, 0.24 nM, and 0.82 microM, respectively. Gypsophilin may be one of the most active RNA N-glycosidases among the RIPs known to date. Immunoelectron microscopic localization of gypsophilin in the leaves shows that the protein is accumulated densely in the intercellular spaces and is also distributed within vacuoles in the cytoplasm.

Mechanism of substrate recognition by the ribotoxin, alpha-sarcin.

The substrate recognition and catalytic mechanisms of alpha-sarcin were explored with kinetic method by using synthetic 25-mer RNA mimicking the alpha-sarcin/ricin loop in 23S rRNA of E. coli ribosomes. The oligomer containing deoxy-G at the site of alpha-sarcin (G14) was a potent competitive inhibitor. The RNA having deoxy-G8 however, increases the Kcat value by about five times but without significant alteration on Km. Surprisingly, the deletion of G8 makes the oligomer become a strong noncompetitive inhibitor of the enzyme. These results suggested that there are at least two sites in the RNA substrate which are recognized by alpha-sarcin, one is the G8 bulge or at around its neighbor and the other is the GAGA in the sarcin/ricin loop of the rRNA.

In vitro selection of aptamers that bind to ribosome-inactivating toxins.

Ribosome-inactivating proteins, such as ricin, pepocin and gypsophilin, catalyze the hydrolysis of a single N-glycosidic bond at a specific position in rRNAs. Aptamers targeting pepocin were selected from a random sequence RNA pool that spanned 30 positions. After 8 rounds, the anti-pepocin aptamers were sequenced and a conserved hairpin motif was identified. Interestingly, the selected motif is quite different from the toxin-binding domains of rRNAs.

Purification, characterization and subcellular localization of a type-1 ribosome-inactivating protein from the sarcocarp of Cucurbita pepo.

The flesh of the fruit of Cucurbita pepo contains a type-1 ribosome-inactivating protein (RIP), which we named pepocin. Pepocin was purified to apparent homogeneity by acid fractionation, ion-exchange chromatography and adsorption chromatography. The protein was found to have a molecular mass of 26 kDa and a pI of about 9.9. It does not contain glycosidic linkages. The protein inhibits protein synthesis in a rabbit-reticulocyte lysate with an IC50 (concentration causing 50% inhibition) of 15.4 pM, and depurinates 28S rRNA in the ribosomes of the lysate in a manner identical to that of ricin A-chain and other RIP. The enzyme is also active on wheat-germ ribosomes and on Escherichia coli ribosomes. The sequence of the N-terminal 20 amino acids of the protein reveals a close relationship to other RIP. Immunoelectron-microscopic localization of pepocin in the sarcocarp shows that the protein is predominantly localized in intercellular spaces. In addition, the immunolocalized signals are observed in leaf intercellular spaces.

Serine tRNA complementary to the nonuniversal serine codon CUG in Candida cylindracea: evolutionary implications.

In the asporogenic yeast Candida cylindracea, the codon CUG is read as serine instead of leucine. This is an unusual instance in which the amino acid assignment of a codon deviates from the universal code. To infer the evolutionary process of this change, the tRNA with the anticodon sequence CAG, which is complementary to and thus responsible for translation of the codon CUG, has been identified. Indeed, this tRNA translates an in-frame CUG codon in a synthetic mRNA as serine in an in vitro translation system. The gene for the tRNA is interrupted by an intron in the anticodon loop. Sequence comparisons of the tRNA and its gene suggest that a single cytidine was inserted into the anticodon loop of the gene for tRNA(Ser)IGA during evolution to produce tRNA(Ser)CAG. The tRNA(Ser)CAG may be produced from its precursor molecule containing the cytidine insertion by splicing.

Construction of yeast tRNA(Tyr) derivatives lacking in large part of the molecule and their tyrosine acceptance.

Derivatives of yeast tRNA(Tyr) lacking in the anticodon-arm (and D-arm) were constructed by a combination of partial digestion with RNase T1 and joining with T4 RNA ligase. Aminoacylation analyses of these derivatives ("3/4 molecule" or "1/2 molecule") showed that sufficient information for binding to TyrRS is contained mainly in the aminoacyl-stem (and T psi C-arm) of yeast tRNA(Tyr), but further information, possibly in the anticodon-arm, is necessary for efficient acceptance of tyrosine.