[A New Aspect of Anti-Glycolipid Antibodies in Guillain-Barré Syndrome: Ca2+-Dependent Antibody in Fisher Syndrome-Related Disorders].

Anti-glycolipid antibodies are key to revealing the pathomechanisms of Guillain-Barré syndrome (GBS). There are correlations between the antigen specificities of the antibodies, clinical features, and preceding infectious agents. It has also been found that some glycoantigens are localized in human peripheral nervous tissues, corresponding to the clinical features. Antibody-detection methods are still evolving. The discovery of antibodies against glycolipid complexes expanded the horizon of anti-glycolipid research in GBS, which had started from isolated antigens. Recently, IgG antibodies against ganglioside GQ1b-related antigens that required Ca2+ cations in the antigen-antibody reaction (Ca2+-dependent anti-GQ antibody) have also been detected in patients with Fisher syndrome, or other related disorders, who were GQ1b-seronegative in conventional assays without adding Ca2+. It is suggested that Ca2+ interacts with disialosyl groups [NeuAc (a2-8) NeuAc (a2-)] in gangliosides, and that the Ca2+-dependent antibodies recognize the Ca2+-bound conformation of GQ1b.

Expression of RSOsPR10 in rice roots is antagonistically regulated by jasmonate/ethylene and salicylic acid via the activator OsERF87 and the repressor OsWRKY76, respectively.

Plant roots play important roles in absorbing water and nutrients, and in tolerance against environmental stresses. Previously, we identified a rice root-specific pathogenesis-related protein (RSOsPR10) induced by drought, salt, and wounding. RSOsPR10 expression is strongly induced by jasmonate (JA)/ethylene (ET), but suppressed by salicylic acid (SA). Here, we analyzed the promoter activity of RSOsPR10. Analyses of transgenic rice lines harboring different-length promoter::ß-glucuronidase (GUS) constructs showed that the 3-kb promoter region is indispensable for JA/ET induction, SA repression, and root-specific expression. In the JA-treated 3K-promoter::GUS line, GUS activity was mainly observed at lateral root primordia. Transient expression in roots using a dual luciferase (LUC) assay with different-length promoter::LUC constructs demonstrated that the novel transcription factor OsERF87 induced 3K-promoter::LUC expression through binding to GCC-cis elements. In contrast, the SA-inducible OsWRKY76 transcription factor strongly repressed the JA-inducible and OsERF87-dependent expression of RSOsPR10. RSOsPR10 was expressed at lower levels in OsWRKY76-overexpressing rice, but at higher levels in OsWRKY76-knockout rice, compared with wild type. These results show that two transcription factors, OsERF87 and OsWRKY76, antagonistically regulate RSOsPR10 expression through binding to the same promoter. This mechanism represents a fine-tuning system to sense the balance between JA/ET and SA signaling in plants under environmental stress.

Ca(2+)-dependent anti-GQ1b antibody in GQ1b-seronegative Fisher syndrome and related disorders.

Although serum IgG anti-ganglioside GQ1b antibody is the most specific biomarker for Fisher syndrome and its related disorders (FS-RD), 10%-30% of the patients are still negative in conventional assays ("GQ1b-seronegative") and the relationship between GQ1b-seropositive and -seronegative patients has been unclear. Some molecules require Ca(2+) cations to interact with their ligands (Ca(2+)-dependency). Here we have investigated whether Ca(2+)-dependency is also present in anti-GQ1b antibodies in FS-RD, especially in the GQ1b-seronegative patients and show that IgG antibodies against GQ1b-related antigens (isolated GQ1b and GQ1b-containing complexes) are detected Ca(2+)-dependently in the majority of GQ1b-seronegative patients with FS-RD. The Ca(2+)-dependent antibodies might react specifically with GQ1b-Ca(2+) conformation. This is the first demonstration of disease-related Ca(2+)-dependent antibodies in neurological field. GQ1b-related pathology would be involved in FS-RD more extensively than previously revealed.

Yucasin is a potent inhibitor of YUCCA, a key enzyme in auxin biosynthesis.

Indole-3-acetic acid (IAA), an auxin plant hormone, is biosynthesized from tryptophan. The indole-3-pyruvic acid (IPyA) pathway, involving the tryptophan aminotransferase TAA1 and YUCCA (YUC) enzymes, was recently found to be a major IAA biosynthetic pathway in Arabidopsis. TAA1 catalyzes the conversion of tryptophan to IPyA, and YUC produces IAA from IPyA. Using a chemical biology approach with maize coleoptiles, we identified 5-(4-chlorophenyl)-4H-1,2,4-triazole-3-thiol (yucasin) as a potent inhibitor of IAA biosynthesis in YUC-expressing coleoptile tips. Enzymatic analysis of recombinant AtYUC1-His suggested that yucasin strongly inhibited YUC1-His activity against the substrate IPyA in a competitive manner. Phenotypic analysis of Arabidopsis YUC1 over-expression lines (35S::YUC1) demonstrated that yucasin acts in IAA biosynthesis catalyzed by YUC. In addition, 35S::YUC1 seedlings showed resistance to yucasin in terms of root growth. A loss-of-function mutant of TAA1, sav3-2, was hypersensitive to yucasin in terms of root growth and hypocotyl elongation of etiolated seedlings. Yucasin combined with the TAA1 inhibitor l-kynurenine acted additively in Arabidopsis seedlings, producing a phenotype similar to yucasin-treated sav3-2 seedlings, indicating the importance of IAA biosynthesis via the IPyA pathway in root growth and leaf vascular development. The present study showed that yucasin is a potent inhibitor of YUC enzymes that offers an effective tool for analyzing the contribution of IAA biosynthesis via the IPyA pathway to plant development and physiological processes.

RSOsPR10 expression in response to environmental stresses is regulated antagonistically by jasmonate/ethylene and salicylic acid signaling pathways in rice roots.

Plant roots play important roles not only in the absorption of water and nutrients, but also in stress tolerance. Previously, we identified RSOsPR10 as a root-specific pathogenesis-related (PR) protein induced by drought and salt treatments in rice. Transcripts and proteins of RSOsPR10 were strongly induced by jasmonate (JA) and the ethylene (ET) precursor 1-aminocyclopropane-1-carboxylic acid (ACC), while salicylic acid (SA) almost completely suppressed these inductions. Immunohistochemical analyses showed that RSOsPR10 strongly accumulated in cortex cells surrounding the vascular system of roots, and this accumulation was also suppressed when SA was applied simultaneously with stress or hormone treatments. In the JA-deficient mutant hebiba, RSOsPR10 expression was up-regulated by NaCl, wounding, drought and exogenous application of JA. This suggested the involvement of a signal transduction pathway that integrates JA and ET signals in plant defense responses. Expression of OsERF1, a transcription factor in the JA/ET pathway, was induced earlier than that of RSOsPR10 after salt, JA and ACC treatments. Simultaneous SA treatment strongly inhibited the induction of RSOsPR10 expression and, to a lesser extent, induction of OsERF1 expression. These results suggest that JA/ET and SA pathways function in the stress-responsive induction of RSOsPR10, and that OsERF1 may be one of the transcriptional factors in the JA/ET pathway.

Asymmetry of shufflon-specific recombination sites in plasmid R64 inhibits recombination between direct sfx sequences.

The shufflon of plasmid R64 consists of four DNA segments separated and flanked by seven sfx recombination sites. Rci-mediated recombination between any inverted sfx sequences causes inversion of the DNA segments independently or in groups. The R64 shufflon selects one of seven pilV genes encoding type IV pilus adhesins, in which the N-terminal region is constant, while the C-terminal regions are variable. The R64 sfx sequences are asymmetric. The sfx central region and right arm sequences are conserved, but left arm sequences are not. Here we constructed a symmetric sfx sequence, in which the sfx left arm sequence was changed to the inverted repeat of the right arm sequence and made artificial shufflon segments carrying symmetric sfx sequences in inverted or direct orientations. The symmetric sfx sequence exhibited the highest inversion frequency in a shufflon segment flanked by two inverted sfx sequences. Rci-dependent deletion of a shufflon segment flanked by two direct symmetric sfx sequences was observed, suggesting that asymmetry of R64 sfx sequences inhibits recombination between direct sfx sequences. In addition, intermolecular recombination between symmetric sfx sequences was also observed. The extra C-terminal domain of Rci was shown to be essential for inversion of the R64 shufflon using asymmetric sfx sequences but not essential for recombination using symmetric sfx sequences, suggesting that the Rci C-terminal segment helps the binding of Rci to asymmetric sfx sequences. Rci protein lacking the C-terminal domain bound to both arms of symmetric sfx sequence but only to the right arm of asymmetric sfx sequence.

Structure and function of the shufflon in plasmid R64.

Conservative site-specific recombination plays key roles in creating biological diversity in prokaryotes. Most site-specific inversion systems consist of two recombination sites and a recombinase gene. In contrast, the shufflon multiple inversion system of plasmid R64 consists of seven sfx recombination sites, which separate four invertible DNA segments, and the rci gene encoding a site-specific recombinase of the integrase family. The rci product mediates recombination between any two inverted sfx sites, resulting in the inversion of four DNA segments independently or in groups. Random shufflon inversions construct seven pilV genes encoding constant N-terminal segment with different C-terminal segments. The pilV products are tip-located adhesins of the type IV pilus, called the thin pilus, of R64 and recognize lipopolysaccharides of recipient bacterial cells during R64 liquid matings. Thus, the shufflon determines the recipient specificity of liquid matings. Rci protein of R64 was overexpressed, purified, and used for in vitro recombination reactions. The cleavage and rejoining of DNA strands in shufflon recombinations were found to take place in the form of a 5' protruding 7-bp staggered cut within sfx sequences. Thus, the sfx sequence is asymmetric: only the 7-bp spacer sequence and the right arm sequence are conserved among various R64 sfxs, whereas the sfx left arm sequences are not conserved. Rci protein was shown to bind to entire sfx sequences, suggesting that it binds to the right arms of the sfx sequences in a sequence-specific manner and to their left arms in a non-sequence-specific manner. The sfx left arm sequences greatly affected the shufflon inversion frequency. The artificial symmetric sfx sequence, in which the sfx left arm was changed to the inverted repeat sequence of the right arm, exhibited the highest inversion frequency. Rci-dependent deletion of a DNA segment flanked by two symmetric sfx sequences in direct orientation was observed, suggesting that the asymmetry of sfx sequences may prevent recombination between sfx sequences in direct orientation in the R64 shufflon. The Rci C-terminal domain was not required for recombination using the symmetric sfx sequence. A model, where the C-terminal domain of Rci protein plays a key role in the sequence-specific and non-specific binding of Rci to asymmetric sfx sites, was proposed. Site-specific recombination in the temperate phage Mx8 of M. xanthus was also described. The Mx8 attP site is located within the coding sequence of the Mx8 intP gene. Therefore, the integration of Mx8 into the M. xanthus chromosome results in the conversion of the intP gene into a new gene, intR. As a result of this conversion, the 112-amino-acid C-terminal sequence of the intP product is replaced with a 13-amino acid sequence of the intR product. The C-terminal domain of Mx8 IntP recombinase is only required for integration and not for excision.

Structure and function of the shufflon in plasmid r64.

Conservative site-specific recombination plays key roles in creating biological diversity in prokaryotes. Most site-specific inversion systems consist of two recombination sites and a recombinase gene. In contrast, the shufflon multiple inversion system of plasmid R64 consists of seven sfx recombination sites, which separate four invertible DNA segments, and the rci gene encoding a site-specific recombinase of the integrase family. The rci product mediates recombination between any two inverted sfx sites, resulting in the inversion of four DNA segments independently or in groups. Random shufflon inversions construct seven pilV genes encoding constant N-terminal segment with different C-terminal segments. The pilV products are tip-located adhesins of the type IV pilus, called the thin pilus, of R64 and recognize lipopolysaccharides of recipient bacterial cells during R64 liquid matings. Thus, the shufflon determines the recipient specificity of liquid matings. Rci protein of R64 was overexpressed, purified, and used for in vitro recombination reactions. The cleavage and rejoining of DNA strands in shufflon recombinations were found to take place in the form of a 5' protruding 7-hp staggered cut within sfx sequences. Thus, the sfx sequence is asymmetric: only the 7-bp spacer sequence and the right arm sequence are conserved among various R64 sfxs, whereas the sfx left arm sequences are not conserved. Rci protein was shown to bind to entire sfx sequences, suggesting that it binds to the right arms of the sfx sequences in a sequence-specific manner and to their left arms in a non-sequence-specific manner. The sfx left arm sequences greatly affected the shufflon inversion frequency. The artificial symmetric sfx sequence, in which the sfx left arm was changed to the inverted repeat sequence of the right arm, exhibited the highest inversion frequency. Rci-dependent deletion of a DNA segment flanked by two symmetric sfx sequences in direct orientation was observed, suggesting that the asymmetry of sfx sequences may prevent recombination between sfx sequences in direct orientation in the R64 shufflon. The Rci C-terminal domain was not required for recombination using the symmetric sfx sequence. A model, where the C-terminal domain of Rci protein plays a key role in the sequence-specific and non-specific binding of Rci to asymmetric sfx sites, was proposed. Site-specific recombination in the temperate phage Mx8 of M. xanthus was also described. The Mx8 attP site is located within the coding sequence of the Mx8 intP gene. Therefore, the integration of Mx8 into the M. xanthus chromosome results in the conversion of the intP gene into a new gene, intP. As a result of this conversion, the 112-amino-acid C-terminal sequence of the intP product is replaced with a 13-amino acid sequence of the intR product. The C-terminal domain of Mx8 IntP recombinase is only required for integration and not for excision.

Sequence-specific and non-specific binding of the Rci protein to the asymmetric recombination sites of the R64 shufflon.

Specific cleavages within the shufflon-specific recombination site of plasmid R64 were detected by primer extension when a DNA fragment carrying the recombination site was incubated with the shufflon-specific Rci recombinase. Rci-dependent cleavages occurred in the form of a 5' protruding 7 bp staggered cut, suggesting that DNA cleavage and rejoining in the shufflon system take place at these positions. As a result, shufflon crossover sites were designated as sfx sequences consisting of a central 7 bp spacer sequence, and left and right 12 bp arms. R64 sfx sequences are unique among various site-specific recombination sites, since only the spacer sequence and the right arm sequence are conserved among various R64 sfxs, whereas the left arm sequence is not conserved and is not related to the right arm sequence. From nuclease protection analyses, Rci protein was shown to bind to entire R64 and artificial sfx sequences, suggesting that one Rci molecule binds to the conserved sfx right arm in a sequence-specific manner and the second to the sfx left arm in a non-specific manner. The sfx left arm sequences as well as the right arm sequences were shown to determine affinity to Rci and subsequently inversion frequency. Asymmetry of the sfx sequence may be the reason why Rci protein acts only on the inverted sfx sequences.