Xanthomonas campestris lipooligosaccharides trigger innate immunity and oxidative burst in Arabidopsis.

Plants lack the adaptive immunity mechanisms of jawed vertebrates, so they rely on innate immune responses to defense themselves from pathogens. The plant immune system perceives the presence of pathogens by recognition of molecules known as pathogen-associated molecular patterns (PAMPs). PAMPs have several common characteristics, including highly conserved structures, essential for the microorganism but absent in host organisms. Plants can specifically recognize PAMPs using a large set of receptors and can respond with appropriate defenses by activating a multicomponent and multilayered response. Lipopolysaccharides (LPSs) and lipooligosaccharides (LOSs) are major components of the cell surface of Gram-negative bacteria with diverse roles in bacterial pathogenesis of animals and plants that include elicitation of host defenses. Little is known on the mechanisms of perception of these molecules by plants and the associated signal transduction pathways that trigger plant immunity.Here we addressed the question whether the defense signaling pathway in Arabidopsis thaliana was triggered by LOS from Xanthomonas campestris pv. campestris (Xcc), using proteomic and transcriptomic approaches. By using affinity capture strategies with immobilized LOS and LC-MS/MS analyses, we identified 8 putative LOS protein ligands. Further investigation of these interactors led to the definition that LOS challenge is able to activate a signal transduction pathway that uses nodal regulators in common with salicylic acid-mediated pathway. Moreover, we proved evidence that Xcc LOS are responsible for oxidative burst in Arabidopsis either in infiltrated or systemic leaves. In addition, gene expression studies highlighted the presence of gene network involved in reactive oxygen species transduction pathway.

CD1A and CD1E gene polymorphisms are associated with susceptibility to multiple sclerosis.

Multiple sclerosis (MS) is thought to be an autoimmune T-cell-mediated disease directed at myelin antigens of the central nervous system. Besides myelin proteins, lipid components of CNS are supposed to play a role as antigens for T cells in MS. CD1 is a family of MHC-like glycoproteins specialized in capturing and presenting a variety of microbial and self lipids and glycolipids to antigen-specific T cells. CD1-restricted T cells specific for gangliosides and sulfatide have been isolated from subjects with MS and in mice with experimental allergic encephalopathy. We genotyped exon 2 of CD1A and CD1E in 205 MS patients and 223 unrelated healthy controls and determined their association with the presence of anti-ganglioside and anti-sulfatide antibodies. CD1E 01-01 is associated with a reduced risk of MS (OR 0.54, p=0.001); CD1A 02-02 (OR 1.99, p=0.012) or CD1E 02-02 (OR 2.45, p=0.000) with an increased risk. The combination of the genotypes CD1A 02-02 and CD1E 02-02 is present in 90.7% of patients but in only 9.4% controls (OR 94.16, p= 0.000). CD1A and CD1E polymorphisms contribute to the polygenic susceptibility to MS. The functional effects of CD1 polymorphisms are unknown, however changes in CD1 alleles may affect numerous immunological functions.

Cross activity of orthologous WRKY transcription factors in wheat and Arabidopsis.

WRKY proteins are transcription factors involved in many plant processes including plant responses to pathogens. Here, the cross activity of TaWRKY78 from the monocot wheat and AtWRKY20 from the dicot Arabidopsis on the cognate promoters of the orthologous PR4-type genes wPR4e and AtHEL of wheat and Arabidopsis, respectively, was investigated. In vitro analysis showed the ability of TaWRKY78 to bind a -17/+80 region of the wPR4e promoter, containing one cis-acting W-box. Moreover, transient expression analysis performed on both TaWRKY78 and AtWRKY20 showed their ability to recognize the cognate cis-acting elements present in the wPR4e and AtHEL promoters, respectively. Finally, this paper provides evidence that both transcription factors are able to cross-regulate the orthologous PR4 genes with an efficiency slightly lower than that exerted on the cognate promoters. The observation that orthologous genes are subjected to similar transcriptional control by orthologous transcription factors demonstrates that the terminal stages of signal transduction pathways leading to defence are conserved and suggests a fundamental role of PR4 genes in plant defence. Moreover, these results corroborate the hypothesis that gene orthology imply similar gene function and that diversification between monocot and dicot has most likely occurred after the specialization of WRKY function.

Antibodies to ganglioside complexes in Guillain-Barré syndrome: clinical correlates, fine specificity and complement activation.

In the Schwann cells and neuronal plasma membranes the gangliosides are organized in clusters forming complexes of gangliosides in the microdomains termed lipid rafts. We investigated frequency, clinical correlates, fine specificity and pro-inflammatory properties of antibodies to ganglioside complexes (GSCs) in a Guillain Barre syndrome (GBS) population. In 63 patients with different GBS variants we performed an ELISA for antibodies to Campylobacter Jejuni (C. jejuni), gangliosides and GSCs. We studied the fine specificity of antibodies to GSCs by immunoabsorption study and performed a complement activation assay. Twenty-seven percent of patients had antibodies to GSCs and 71 percent had antibodies either to single gangliosides or to GSCs. Patients with antibodies to GSCs had more frequent involvement of cranial nerves but did not present more frequent antecedent respiratory, gastrointestinal or C. jejuni infection, did not have a preferential demyelinating or primary axonal GBS variant and did not develop greater disability at six months. The absorption study showed in 2 sera that antibodies to the complex GD1a/GD1b did not react with the gangliosides forming the complex or other single gangliosides, suggesting that antibodies to GSCs are targeted to new conformational glycoepitopes different from the ones displayed by the single gangliosides. Antibody anti-GSCs activated the complement more frequently than antibodies to single gangliosides. Complement activation indicates that antibodies to GSCs have high avidity, pro-inflammatory properties and may exert a pathogenic role in GBS.

T cell response in acute motor axonal neuropathy.

There is evidence that in the acute axonal motor neuropathy (AMAN) subtype of Guillain-Barré syndrome antibodies to gangliosides, produced through molecular mimicry by antecedent Campylobacter jejuni (C. jejuni) infection, attack gangliosides expressed in human peripheral nerve axolemma, inducing a primary axonal damage. The aim of this study is to investigate whether the T cell response has a role in AMAN pathogenesis. We isolated monocytes from 4 healthy subjects and 5 AMAN patients with antecedent C. jejuni infection and antibodies to GM1 and/or GD1a gangliosides. Immature dendritic cells expressing CD1 molecules cultured with autologous T cells were stimulated with 2 lipopolysaccharides (LPSs) extracted from C. jejuni strains containing GM1 and GD1a-like structures and with GM1 and GD1a. The T cell response to LPSs and to gangliosides was determined by measuring the release of IFN-gamma and TNF-alpha. We observed a T cell response to both LPSs in controls and AMAN patients, whereas only AMAN patients showed T cell reactivity to gangliosides GM1 and GD1a with a tight correlation between T cell reactivity to the ganglioside and individual antibody responses to the same ganglioside. T cells responding to gangliosides were CD1c-restricted CD8 positive and CD27 negative. These findings indicate a contribution of cellular immunity in the pathogenesis of AMAN. A possible role for ganglioside-reactive T cells might be to facilitate the production of antibodies against gangliosides.

Isolated shoulder palsy due to cortical infarction: localisation and electrophysiological correlates of recovery.

The corticospinal tract influences the distal musculature more than the proximal, and the mechanisms involved in recovery of proximal muscle strength after stroke are unclear. A 65 year old man developed right shoulder weakness due to infarction in the left precentral gyrus. MRI showed a 3 mm cortical-subcortical ischaemic lesion in the superior genu of the left precentral gyrus medially to the knob-like structure corresponding to the motor area of the hand. Two months after stroke, when the patient was able to abduct the right arm against gravity and seven months after stroke when the patient had almost completely recovered, maximal TMS of the contralateral and ipsilateral motor cortex during voluntary contraction did not evoke a MEP in the right deltoid either with a focal or a non-focal coil. Recovery of proximal muscles in these cases may be mediated by elements other than the fast corticospinal neurones responsible for MEP generation.

CD1a and CD1e allele frequencies in an Italian population from the Abruzzo region.

CD1 is a small family comprising 5 MHC-like genes located on chromosome 1 and encoding glycoproteins termed CD1a, CD1b, CD1c, CD1d and CD1e. They are expressed mainly on the surface of dendritic cells, monocytes and some thymocytes and are specialized in presenting lipid antigens to T lymphocytes. The structure is similar to that of MHC class I molecules with 3 globular domains and the Beta2-microglobulin. It has been shown that all five human CD1 genes exhibit a limited number of polymorphisms in the alpha1 domain whose effects are still unknown. CD1e results to be the most polymorphic isoform with six CD1e alleles (01, 02 in exon 2 and 03, 04, 05, 06 in ex3) described to date. At this moment, few investigations on the allele frequencies of the CD1 genes have been reported and all additional information improves our knowledge on this new class of antigen-presenting molecules. In order to study possible allelic variations of exon 2 of human CD1a and CD1e genes, we analyzed, by a sensitive technique, the sequence-based typing (SBT), 114 DNA samples from unrelated healthy Italian individuals from the Abruzzo region. Our experimental findings indicate that the allele frequency distribution of both CD1a and CD1e genes is in accordance with that observed in other geographic areas and did not identify any new allele, thus confirming a very low polymorphism.

Decreased integrin gene expression in patients with MS responding to interferon-beta treatment.

Interferon-beta (IFN-beta) ameliorates disease course in a subset of patients with MS. The reasons for heterogeneity of clinical responses, however, are unclear. We assessed possible effects of IFN-beta on the gene expression of the leukocyte adhesion molecules VLA-4 and LFA-1 during the first year of treatment of 50 patients with relapsing-remitting MS who showed differential clinical responses. We observed a significant reduction of VLA-4 (P=0.002) and LFA-1 (P=0.03) mRNA expression compared to baseline in first-year clinical responders (n=22). In contrast, first-year IFN-beta non-responders (n=28) had unchanged levels of VLA-4 and LFA-1. In vitro treatment of PBMC with IFN-beta indicated a direct effect on transcription of the integrins' genes. Transcriptional downmodulation of adhesion molecules during IFN-beta treatment may contribute to its mode of action in MS.

Inter-nerves and intra-nerve conduction heterogeneity in CMTX with Arg(15)Gln mutation.

OBJECTIVE: In X-linked Charcot-Marie-Tooth disease (CMTX), electrophysiological and histopathological studies have suggested either a demyelinating or an axonal polyneuropathy. We report a CMTX family with a striking heterogeneity of nerve conductions between and within nerves. METHODS: Two men and one woman have been studied by conduction velocities, sural nerve biopsy with morphometry (one man) and DNA analysis. RESULTS: In both men motor conduction velocities were slowed in the demyelinating range, conduction velocity differences among nerves in the same subject varied from 13 to 24 m/s, and distal median compound muscle action potential (CMAP) amplitudes were 3-5 times reduced compared to ulnar CMAPs. Abnormal area reduction or excessive temporal dispersion of proximal CMAP was present in at least two nerves in all patients. Sural nerve biopsy showed reduction of large myelinated fibres, cluster formations, occasional onion bulbs. Teased fibres study revealed short internodes for fibre diameter, enlarged Ranvier nodes but no evidence of segmental demyelination and remyelination. DNA analysis showed an Arg(15)Gln mutation in connexin32 gene in all patients. CONCLUSIONS: In this family conduction slowing and segmental conduction abnormalities, in absence of morphological evidence of de-remyelination, may be related to short internodes, widened Ranvier nodes and the specific effect of the mutation. The occurrence in some CMTX patients of a non uniform involvement between and within nerves, as in acquired demyelinating neuropathies, should be kept in mind to avoid misdiagnoses.

Acute motor conduction block neuropathy Another Guillain-Barré syndrome variant.

OBJECTIVE: To report two patients with an acute exclusively motor neuropathy with conduction blocks. METHODS: Serial electrophysiologic studies were carried out. RESULTS: Two patients developed symmetric proximal and distal weakness without sensory abnormalities after enteritis. Tendon reflexes were normal in one patient and brisk in the other. One patient had high titer immunoglobulin G to GD1a and GM1, and the other to GD1b, GD1a, and GM1 and a recent Campylobacter jejuni infection. Electrophysiology showed early partial motor conduction block in intermediate and distal nerve segments, normal sensory conductions even across the sites of conduction block, and normal somatosensory evoked potentials. Conduction blocks resolved in 2 to 5 weeks without excessive temporal dispersion of proximal motor responses. CONCLUSIONS: Acute motor neuropathy with normal or brisk tendon reflexes, conduction block, and fast recovery appears to be a variant of Guillain-Barré syndrome. Conduction block may result from immune-mediated conduction failure at the nodes of Ranvier without demyelination.

Recombinant wheat antifungal PR4 proteins expressed in Escherichia coli.

PR proteins are soluble and host-coded molecules with antifungal activity induced by a variety of agents. Wheat contains several PR proteins and among them are those of the class 4 coded wheatwin1 and wheatwin2; the two native proteins have been isolated from wheat kernel and the coding cDNA clones have been recently characterized. Herein, we report the expression of recombinant wheatwin1 and wheatwin2 in Escherichia coli-insoluble fractions; a new protocol for the purification in high yields and correct processing of the two proteins was developed. The recombinant proteins have molecular weights identical to that of the native proteins, indicating that the removal of the N-terminal methionine and cyclization of glutamine to pyroglutamate was complete. Both recombinant proteins inhibited in vitro the growth of Fusarium culmorum exhibiting antifungal properties similar to those of the native proteins.

Isolation and amino acid sequence of two new PR-4 proteins from wheat.

We have purified and characterized two new pathogenesis-related (PR) proteins from wheat belonging to the PR-4 family. We named the proteins wheatwin3 and wheatwin4 in analogy with the previously characterized wheatwin1 and wheatwin2. Their isoelectric points were 7.1 and 8.4, respectively. We determined the complete amino acid sequence of both proteins by a rapid approach based on the knowledge of the primary structures of the homologous wheatwin1 and wheatwin2. Wheatwin3 differs from wheatwin1 in one substitution at position 88, while wheatwin4 differs from wheatwin2 in one substitution at position 78. The secondary structure and solvent accessibility of these residues were determined on the three-dimensional model of wheatwinl. Residue 88 was very accessible and was located in a flexible region. Preliminary results indicate that, like wheatwin1 and wheatwin2, wheatwin3 and wheatwin4 have antifungal activity.

A basic peroxidase from wheat kernel with antifungal activity.

A basic heme-peroxidase (WP1) was purified to homogeneity from wheat (Triticum aestivum) kernels. The protein was not glycosylated and exhibited a molecular mass of 36 kDa and a pI of 8.0. The N-terminal amino acid sequence revealed a very high similarity with a wheat flour peroxidase allergen associated with baker's asthma. WPI showed indole-3-acetic acid oxidase activity in the presence of Mn2+ and phenolic cofactors. Antifungal assays performed in vitro towards phytopathogenic fungi indicated that WP1 was active in inhibiting germ tube elongation. This first report on antifungal properties of a heme-peroxidase gives experimental support to the idea that peroxidases play a defensive role against invading pathogens.

Low-dose oral methotrexate treatment in chronic progressive multiple sclerosis.

We aimed to further assess the safety and efficacy of low-dose oral methotrexate (LDOM) treatment for chronic progressive MS (CPMS). We studied 20 CPMS patients, including 16 with secondary progressive MS who had shown disease progression in the previous year. The mean follow-up was 23 months. The mean EDSS score was 6.3+/-1.1 before treatment and 6.4+/-1.1 after one year of treatment. At one year, 15 of 20 patients were still being treated, and 10 were stable. Twelve patients have completed 18 months of treatment, and eight are stable. Two patients stopped treatment because of side effects, two more because they did not perceive benefit, and one was lost to follow-up. Six patients had mild, transient increases in liver enzymes not requiring treatment interruption, and two had localized herpes zoster. Magnetic resonance imaging (MRI) performed before treatment and at one year remained unchanged in responders. We confirm that LDOM is safe in carefully selected and monitored CPMS patients. MTX is inexpensive and, given its anti-inflammatory and immunomodulatory properties, may be used as add-on therapy in non-responders to interferon beta, although hepatic toxicity may be a problem in long-term treatment.

Formation of the catecholamine release-inhibitory peptide catestatin from chromogranin A. Determination of proteolytic cleavage sites in hormone storage granules.

The catestatin fragment of chromogranin A is an inhibitor of catecholamine release, but its occurrence in vivo has not yet been verified, nor have its precise cleavage sites been established. Here we found extensive processing of catestatin in chromogranin A, as judged by catestatin radioimmunoassay of size-fractionated chromaffin granules. On mass spectrometry, a major catestatin form was bovine chromogranin A(332-364); identity of the peptide was confirmed by diagnostic Met(346) oxidation. Further analysis revealed two additional forms: bovine chromogranin A(333-364) and A(343-362). Synthetic longer (chromogranin A(332-364)) and shorter (chromogranin A(344-364)) versions of catestatin each inhibited catecholamine release from chromaffin cells, with superior potency for the shorter version (IC(50) approximately 2.01 versus approximately 0.35 microm). Radioimmunoassay demonstrated catestatin release from the regulated secretory pathway in chromaffin cells. Human catestatin was cleaved in pheochromocytoma chromaffin granules, with the major form, human chromogranin A(340-372), bounded by dibasic sites. We conclude that catestatin is cleaved extensively in vivo, and the peptide is released by exocytosis. In chromaffin granules, the major form of catestatin is cleaved at dibasic sites, while smaller carboxyl-terminal forms also occur. Knowledge of cleavage sites of catestatin from chromogranin A may provide a useful starting point in analysis of the relationship between structure and function for this peptide.

Probing the modelled structure of wheatwin1 by controlled proteolysis and sequence analysis of unfractionated digestion mixtures.

We set up a method to get rapid information on the three-dimensional structure of peptide and proteins of known sequence. Both native and alkylated polypeptide is hydrolyzed with a number of proteases at different digestion times and the resulting mixtures are compared by HPLC analysis to establish the differences in the hydrolysis pathways of the folded and unfolded molecule. Then, the unfractionated digestion mixtures of the native polypeptide are submitted to automatic sequence analysis to identify the hydrolysis sites. The sequence of each fragment present in the mixtures is reconstructed and its amount determined by quantitative data of the sequence analyses. We used this approach to determine the amino acid surface accessibility of wheatwin1, a pathogenesis-related protein from wheat, and constructed a predictive three-dimensional model based on the knowledge of the tertiary structure of barwin, a highly homologous protein from barley. The procedure allowed us to quickly identify and quantify the hydrolysis at the susceptible bonds which could be classified as exposed, partially hidden, or inaccessible. The results were useful to evidentiate and discuss concordances and differences between experimental and model predicted accessibilities of amino acid residues. Proteins 1999;36:192-204.

Isolation and characterisation of wheat cDNA clones encoding PR4 proteins.

Two cDNA clones encoding the previously characterised PR4 proteins wheatwin 1 and wheatwin2 from wheat (Triticum aestivum cv. S. Pastore) have been identified and named wPR4a and wPR4b, respectively. The clones have been isolated by screening a cDNA library with a specific cDNA probe obtained by RT-PCR. The wPR4a and wPR4b cDNAs contain open reading frames of 441 and 447 bp that encode for wheatwin1 and wheatwin2, respectively.

Laccase from the white-rot fungus Trametes trogii.

The white-rot fungus Trametes trogii excretes a main laccase showing a molecular mass of 70 kDa, acidic isoelectric point and N-terminal sequence homologous to that of several phenol oxidases. The purified enzyme oxidizes a number of phenolic and non-phenolic compounds; recalcitrant molecules may be converted into substrates by introducing, in the correct position, o- or p-orienting ring-activating groups.